Methods for the administration of amifostine and related compounds

ABSTRACT

The present invention provides methods of administering aminoalkyl phosphorothioate and/or aminoalkyl thiol compounds to patients receiving radiation therapy in a manner that significantly reduces or decreases the adverse or undesirable side-effects of the compounds as compared with conventional intravenous administration.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of application Ser.No. 09/586,753, filed Jun. 5, 2000 now U.S. Pat. No. 6,218,377, nowallowed, which is a continuation of application Ser. No. 09/021,139,filed Feb. 10, 1998, now U.S. Pat. No. 6,127,351, issued Oct. 3, 2000,which is a continuation-in-part of application Ser. No. 08/798,840,filed Feb. 12, 1997, now U.S. Pat. No. 6,051,563, issued Apr. 18, 2000,each of which are incorporated by reference herein in its entirety.

1. INTRODUCTION

The present invention relates to methods of administering aminoalkylphosphorothioate and/or aminoalkyl thiol compounds to a subject in amanner that reduces or decreases the undesirable side effects of thecompounds. One aspect of the invention relates to the subcutaneousadministration of amifostine and/or its active metabolite to a patient,which reduces adverse side effects. Another aspect of the inventionrelates to methods of administering amifostine and/or its activemetabolite to a patient in a manner such that a characteristicpharmacokinetic profile is obtained. When administered according to thecharacteristic pharmacokinetic profile, fewer adverse side effects areexperienced by patients.

2. BACKGROUND OF THE INVENTION

Amifostine (also known as WR-2721) has been shown to be useful as aradiation protectant in cancer patients receiving radiation therapy(Constine et al., 1986, “Protection by WR-2721 of Human Bone MarrowFunction Following Irradiation” Int. J. Radia. Oncol. Biol. Phys.12:1505-8; Liu et al., 1992, “Use of Radiation with or Without WR-2721in Advanced Rectal Cancer” Cancer 69(11):2820-5; Wadler et al., 1993,“Pilot Trial of Cisplatin, Radiation and WR-2721 in Carcinoma of theUterine Cervix: A New York Gynecologic Oncology Group Study” J. Clin.Oncol. 11(8):1511-6; Buntzel et al., 1996, “Selective Cytoprotectionwith Amifostine in Simultaneous Radiochemotherapy of Head Neck Cancer”Ann. Oncol. 7(Suppl.5):81(381P)). Amifostine is a pro-drug that isdephosphorylated at the tissue site by alkaline phosphatase to the freethiol, which is the active metabolite (also known as WR-1065). Onceinside the cell, the active free thiol can protect against thetoxicities associated with radiation by acting as a scavenger for oxygenfree-radicals that are produced by ionizing radiation (Yuhas, 1977, “Onthe Potential Application of Radioprotective Drugs in Solid TumorRadiotherapy,” In: Radiation-Drug Interactions in Cancer Management pp.303-52; Yuhas, 1973, “Radiotherapy of Experimental Lung Tumors in thePresence and Absence of a Radioprotective Drug S-2-(3-Aminopropylamino)thylphosphorothioc Acid (WR-2721)” J. Natl. Cancer Inst. 50:69-78;Philips et al., 1984, “Promise of Radiosensitizers and Radioprotectorsin the Treatment of Human Cancer” Cancer Treat. Rep. 68:291-302).

Amifostine's ability to selectively protect normal tissues is based onthe differential metabolism and uptake of amifostine into normal tissueversus tumor tissue. Amifostine is rapidly taken up and retained innormal tissues. Differences in capillary and membrane-bound alkalinephosphatase concentration and pH between normal and tumor tissues havebeen shown to favor the conversion of the pro-drug and uptake of theactive form of amifostine, the free thiol, into normal tissues. Coupledwith the fact that normal cells concentrate the free thiol at a fasterrate than tumors and retain it for longer periods of time, amifostine isable to selectively protect normal tissues against the toxicitiesassociated with radiation without negatively affecting the antitumorresponse. The marked differences in tissue uptake and retention betweennormal and tumor tissues produces a temporary state of acquired drugresistance in normal tissues, analogous to that produced by an excess ofendogenous glutathione.

For a cytoprotector to be useful in radiation therapy, the compound mustbe tolerated on a daily basis, up to 4 or 5 days a week for severalweeks, prior to the delivery of conventional doses of radiation.McDonald et al. (McDonald, 1994, “Preliminary Results of a Pilot StudyUsing WR-2721 Before Fractionated Irradiation of Head and Neck to ReduceSalivary Gland Dysfunction” Int. J. Radiat. Oncol. Biol. Phys.29(4):747-54; McDonald et al., 1995, “Amifostine Preserves the SalivaryGland Function During Irradiation of the Head and Neck” Eur. J. Cancer31a(Supp. 5):415) have conducted a dose-escalation study of amifostineand radiation in patients with head and neck cancer. These resultssuggest that daily administration of amifostine (200 mg/m² via a6-minute intravenous infusion) prior to radiation protects the salivarygland against the toxicities of radiation.

Amifostine has also been shown to stimulate bone marrow growth, and iscurrently in Phase II clinical trials as a bone marrow stimulant inpatients suffering from myelodysplastic syndrome (List et al., 1996,“Amifostine Promotes Multilineage Hematopoiesis in Patients withMyelodysplastic Syndrome (MDS): Results of a Phase I/II Clinical Trial”Am. J. Hem. 1 (Abstract); List et al., 1996, “Amifostine Promotes invitro and in vivo Hematopoiesis in Myelodysplastic Syndromes” Chem.Found Sympos. (Abstract); List et al., 1996, “Amifostine PromotesMultilineage Hematopoiesis in Patients with Myelodysplastic Syndrome(MDS): Results of a Phase I/II Clinical Trial,” Abstract, 8th AnnualMeeting, American Society of Hematology, Orlando, Fla.). In this study,amifostine is being administered via intravenous infusion.

Intravenous administration of amifostine suffers from several seriousdrawbacks. First, administering compounds intravenously is extremelyinconvenient, particularly when a daily dosing schedule for severalweeks, or potentially several months in the case of MDS, is necessary,requiring a skilled practitioner to administer the dose. Second, whenadministered intravenously, patients suffer from dose-dependentundesirable side-effects such as nausea, vomiting, emesis andhypotension, as well as flushing or feeling of warmth, chills or feelingof coldness, dizziness, somnolence, hiccups and sneezing. A decrease inserum calcium concentration is a known pharmacological effect ofintravenously administered amifostine. Allergic reactions ranging frommild skin rashes to rigors have also rarely occurred in conjunction withintravenously administered amifostine. At present, there are no knownmethods, other than co-administering agents such as anti-emetics, ofreducing or avoiding these undesirable side effects. Third, there arerelated costs associated with intravenous administration, includingpersonnel, equipment and medical measures to attenuate side effects.

The human pharmacokinetic profile of amifostine has been investigated incancer patients following a single intravenous bolus dose (150 mg/kg)(Shaw et al., 1986, “Human Pharmacokinetics of WR-2721” Int. J. Radiat.Oncol. Biol. Phys. 12:1501-4), a single 15-minute intravenous infusion(up to 910 mg/m²) (Shaw et al., 1988, “Pharmacokinetics of WR-2721”Pharmac. Ther. 39:195-201; Shaw et al., 1994, “Pharmacokinetics ofAmifostine in Cancer Patients: Evidence for Saturable Metabolism” Proc.Amer. Cos. Clin. Oncol. 13:144; U.S. Bioscience, 1994, “Pharmacokineticsof Single ose Amifostine (WR-2721; Ethyol)” ETH PK 3) and repeatedinfusions (up to 910 mg/M² per dose) (U.S. Bioscience, 1994,“Pharmacokinetics of Double Dose Amifostine (WR-2721; Ethyol) ithCorresponding Measurements of WR-1065 in Plasma and Bone arrow Cells”ETH PK 4). These studies showed that mifostine is rapidly cleared fromthe plasma with a distribution half-life of less than 1 minute and anelimination half-life of approximately 9 minutes. Less than 10% ofamifostine remained in the plasma 6 minutes after intravenousadministration. No previous human clinical harmacokinetic studies havebeen conducted using either orally or subcutaneously administeredamifostine.

Tabachnik reported that the oral administration of amifostine reducedsputum viscosity in cystic fibrosis patients (Tabachnik et al., 1980,“Studies on the Reduction of Sputum Viscosity in Cystic Fibrosis Usingan Orally Absorbed Protected Thiol.” J. Pharm. Exp. Ther. 214:246-9;Tabachnik et al., 1982, “Protein Binding of N-2-Mercaptoethyl-13-Diaminopropane via Mixed Disulfide Formation After Oral Administrationof WR-2721” J. Pharm Exp. Ther. 220:243-6). However, these studies didnot demonstrate that this mode of administration reduced adverse sideeffects commonly associated with intravenously administered amifostine.Furthermore, a study of the pharmacokinetic profile of the admnisteredcompounds was not conducted in these patients.

3. SUMMARY OF THE INVENTION

The present invention provides methods of administering aminoalkylphosphorothioate and/or aminoalkyl thiol compounds to a subject,including humans, in a manner which decreases or reduces the undesirableside effects of the compounds. One aspect of the invention relates tothe subcutaneous administration of an aminoalkyl phosphorothioate and/oraminoalkyl thiol compound to a human subject. Another aspect of theinvention relates to the administration of an aminoalkylphosphorothioate and/or aminoalkyl thiol compound to a patient in amanner such that a characteristic pharmacokinetic profile is obtained.The pharmacokinetic profile is generally characterized by a first regionwherein the plasma and/or a whole blood concentration of administeredcompound, an active metabolite thereof, or both gradually increases to amaximum concentration, a second region wherein the maximum plasma and/orwhole blood concentration is substantially maintained, or plateaus, anda third region wherein the plasma and/or whole blood concentrationgradually decreases to baseline levels. The rate at which the plasmaand/or whole blood concentration of the administered compound (and/or anactive metabolite thereof) increases to the maximum level is markedlyslower than that achieved with conventional intravenous administration.

Administering aminoalkyl phosphorothioate and/or aminoalkyl thiolcompounds according to the methods of the invention significantlyreduces or decreases the adverse or undesirable side effects suffered bypatients as compared to conventional intravenous administration, withoutsubstantially affecting the efficacy of the applied dose. Thus, themethods of the invention can be advantageously used in conjunction withtreatment strategies for delivering aminoalkyl phosphorothioate and/oraminoalkyl thiol compounds to patients without inducing the vomiting,nausea, emesis, hypotension or other undesirable side-effects, includingbut not limited to, flushing or feeling of warmth, chills or feeling ofcoldness, dizziness, somnolence, hiccups, sneezing, decreased serumcalcium levels and allergic reactions that are commonly experienced withconventional intravenous administration.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the average whole blood concentration-time curvesfor amifostine (parent drug WR-2721 plus active metabolite WR-1065)following a 500 mg subcutaneous injection, a 500 mg oral solution and a200 mg/m² intravenous infusion (over 7.5 minutes) to 12 subjects, 0 to240 minutes after drug administration (——iv; ---sc; — —oral).

FIG. 2 is a graph of the average plasma concentration-time curves foramifostine (parent drug WR-2721) following a 500 mg subcutaneousinjection, and a 200 mg/M² intravenous infusion (over 7.5 minutes) to 12subjects, 0 to 240 minutes after drug administration (——iv; ---sc).

FIG. 3 is a graph of the average serum concentration-time curves foractive metabolite (WR-1065) following a 500 mg subcutaneous injection, a500 mg oral solution and a 200 mg/m² intravenous infusion (over 7.5minutes) to 12 subjects, 0 to 240 minutes after drug administration(——iv; ---sc; — —oral)

FIGS. 4A and 4B are two graphs of the body weight of animals treatedwith amifostine or saline and a single dose of irradiation at 16.5 Gy.Amifostine and saline were administered by i.p. and s.c. routes. Controlanimals received saline without irradiation. Amifostine was dissolved at500 mg in 9.7 ml of 0.9% Nacl to achieve a final concentration of 50mg/ml. About 0.1-0.2 ml of amifostine was injected in each animal basedon the body weight. Amifostine was administered at a dose of 400 mg/kgbody weight (FIG. 4A) and at a dose of 200 mg/kg body weight (FIG. 4B).

FIGS. 5A and 5B are two graphs of the mucosal erythema scores of animalstreated with amifostine or saline and a single dose of irradiation at16.5 Gy. Amifostine and saline were administered by i.p. and s.c.routes. Control animals received saline without irradiation. Amifostinewas dissolved at 500 mg in 9.7 ml of 0.9% Nacl to achieve a finalconcentration of 50 mg/ml. About 0.1-0.2 ml of amifostine was injectedin each animal based on the body weight. Amifostine was administered ata dose of 400 mg/kg body weight (FIG. 5A) and at a dose of 200 mg/kgbody weight (FIG. 5B).

FIGS. 6A and 6B are two graphs of the mucosal edema scores of animalstreated with amifostine or saline and a single dose of irradiation at16.5 Gy. Amifostine and saline were administered by i.p. and s.c.routes. Control animals received saline without irradiation. Amifostinewas dissolved at 500 mg in 9.7 ml of 0.9% Nacl to achieve a finalconcentration of 50 mg/ml. About 0.1-0.2 ml of amifostine was injectedin each animal based on the body weight. Amifostine was administered ata dose of 400 mg/kg body weight (FIG. 6A) and at a dose of 200 mg/kgbody weight (FIG. 6B).

FIGS. 7A-7C are three graphs of colony formation by bone marrowprogenitor cells obtained from MDS patients after receiving amifostinesubcutaneously.

FIG. 7A shows the formation of CFU-GEMM colonies,

FIG. 7B shows the formation of BFU-E colonies and

FIG. 7C shows the formation of CFU-GM colonies.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of administering aminoalkylphosphorothioate and/or aminoalkyl thiol compounds to patients in amanner which decreases the undesirable side effects of the compounds ascompared to conventional intravenous administration. The invention isbased, in part, on the quite unexpected discovery that human patientsreceiving subcutaneously administered amifostine experiencedsignificantly fewer incidences of nausea, vomiting, headache,hypotension, lightheadness, somnolence and other undesirable sideeffects commonly associated with conventional intravenous administrationof amifostine than did patients who received amifostine via conventionalintravenous infusion. These side effects coincide with the “spike” or“burst” phase of the pharmacokinetic profile of intravenouslyadministered drug.

The invention is further based, in part, on the observation that thepharmacokinetic profiles of amifostine and its active metabolite WR-1065for intravenously and subcutaneously administered amifostine aresignificantly different. Whereas whole blood concentrations of bothamifostine and its active metabolite WR-1065 peaked and declined rapidlywithin the first 10 minutes following intravenous administration ofamifostine, whole blood concentrations of both of these compoundsincreased at a markedly slower rate following subcutaneous amifostineadministration, reaching maximum and sustained concentration about 15-45minutes after administration.

While not intending to be bound by theory, it is believed that thedecrease in undesirable side effects observed for subcutaneouslyadministered amifostine as compared to intravenously administeredamifostine is in part due to the characteristic pharmacokinetic profileassociated with subcutaneous administration. Thus, in the methods of theinvention a therapeutically effective amount of an aminoalkylphosphorothioate and/or aminoalkyl thiol compound is administered to apatient in a manner such that a characteristic pharmacokinetic profilefor either the administered compound and/or an active metabolite thereofis obtained, thereby significantly reducing the undesirable side effectssuffered by patients receiving such therapy as compared withconventional intravenous administration.

5.1 The Compounds

Compounds which can be advantageously administered according to themethods described herein are aminoalkyl phosphorothioate or aminoalkylthiol compounds which exhibit selective radioprotection orchemoprotection of normal tissues, and/or bone marrow stimulating orhealing activities, and which are suitable for human use with minimaltoxicity. Such aminoalkyl phosphorothioate or aminoalkyl thiolcompounds, as well as pharmaceutically acceptable addition salts and/orhydrates thereof, are either known to those of skill in the art or canbe identified without undue experimentation using established testsroutinely employed in the art.

In one illustrative embodiment, compounds that can be advantageouslyadministered via the methods of the invention are compounds having theformula:

R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I)

or pharmaceutically acceptable addition salts or hydrates thereof,wherein:

R₁ is hydrogen, C₁-C₇ aryl, C₁-C₇ acyl or C₁-C₇ alkyl;

R₂ is hydrogen or PO₃H₂;

n is an integer from 2 to 6; and

m is an integer from 2 to 6.

All of the compounds of formula (I) may be prepared by methods known inthe art (see, e.g., Cortese, 1943, Organic Synthesis pp. 91-93, Coll.Vol. II, Blatt, Ed., John Wiley & Sons, Inc., New York, N.Y.; Akerfeldt,1960, Acta Chem. Scand. 14:1980; Piper et al., 1966, Chem. Ind.(London):2010). Certain aminoalkyl phosphorothioate compounds accordingto formula (I), as well as methods of synthesizing such compounds, aredescribed in detail in U.S. Pat. No. 3,892,824 and WO 96/25045, each ofwhich is incorporated herein by reference in its entirety.

The aminoalkyl phosphorothioate and/or aminoalkyl thiol compounds usefulin the methods of the invention may be in the form of free acids, freebases, or pharmaceutically acceptable addition salts thereof. Such saltscan be readily prepared by treating an aminoalkyl phosphorothioate oraminoalkyl thiol compound with an appropriate acid. Such acids include,by way of example and not limitation, inorganic acids such as hydrohalicacids (hydrochloric, hydrobromic, hydrofluoric, etc.), sulfuric acid,nitric acid, phosphoric acid, etc. and organic acids such as aceticacid, propanoic acid, 2-hydroxyacetic acid, 2-hydroxypropanoic acid,2-oxopropanoic acid, propandioic acid, butandioic acid, etc. Conversely,the salt can be converted into the free base form by treatment withalkali.

The aminoalkyl phosphorothioate and/or aminoalkyl thiol compounds usefulin the methods of the invention, as well as the pharmaceuticallyacceptable addition salts thereof, may be in a hydrated or unhydratedform. Methods of preparing such forms will be apparent to those of skillin the art of organic chemistry.

In a preferred embodiment of the invention, the compounds are those offormula (I), or pharmaceutically acceptable addition salts or hydratesthereof, wherein:

R₁ is hydrogen or methyl;

R₂ is hydrogen or PO₃H₂;

n is 3; and

m is 2 or 3.

In a particularly preferred embodiment of the invention, the compound isS-2-(3-aminopropylamino)ethyl dihydrogen phosphorothioate,H₂N(CH₂)₃NH(CH₂)₂SPO₃H₂ (amifostine or WR-2721), particularly the mono-and tri-hydrates thereof; 2-[3-aminopropylamino]ethanethiol,H₂N(CH₂)₃NH(CH₂)₂SH (the active metabolite of amifostine or WR-1065);S-3-(3-methylamino-propylamino)propyl dihydrogen phosphorothioate,CH₃NH(CH₂)₃NH(CH₂)₃SPO₃H₂ (WR-151327); or S-3-(3-methyl-aminopropylaminopropane thiol, CH₃NH(CH₂)₃NH(CH₂)₃SH(WR-151326).

5.2 Pharmacokinetic Profile

Analysis of the pharmacokinetic profiles of amifostine and its activemetabolite WR-1065 following intravenous and subcutaneous administrationof amifostine reveals several striking differences. Referring now toFIGS. 1-3, the pharmacokinetic profiles of amifostine (FIG. 2), anactive metabolite of amifostine (WR-1065) (FIG. 3) and amifostine andWR-1065 combined (FIG. 1) obtained after intravenous administration ofamifostine (200 mg/M² drug infused over 7.5 min.) are characterized byan initial plasma concentration “spike” or “burst” within the first 20minutes following administration, a time-frame that coincides with theside effect observed clinically: infused drug and/or its activemetabolite is rapidly taken up within the first minute ofadministration, reaching a maximum whole blood concentrationapproximately 8-10 minutes after administration, followed by a rapid(approx. 5-10 fold) decrease in concentration about 10-20 minutes afteradministration. Following this initial concentration “spike” or “burst,”blood levels gradually decrease to zero.

Similar pharmacokinetic profiles were observed with amifostine in cancerpatients following a single intravenous bolus dose (150 mg/M²) (Shaw etal., 1986, “Human Pharmacokinetics of WR-2721” Int. J. Radiat. Oncol.Biol. Phys. 12:1501-4), a single 15-minute intravenous infusion (up to910 mg/M²) (Shaw et al., 1988, “Pharmacokinetics of WR-2721” Pharmac.Ther. 39:195-201; Shaw et al., 1994, “Pharmacokinetics of Amifostine inCancer Patients: Evidence for Saturable Metabolism” Proc. Amer. Cos.Clin. Oncol. 13:144; U.S. Bioscience, 1994, “Pharmacokinetics of SingleDose Amifostine (WR-2721; Ethyol)” ETH PK 3) and repeated infusions (upto 910 mg/m² per dose) (U.S. Bioscience, 1994, “Pharmacokinetics ofDouble Dose Amifostine (WR-2721; Ethyol) with Corresponding Measurementsof WR-1065 in Plasma and Bone Marrow Cells” ETH PK 4). In these studies,amifostine was rapidly cleared from the blood, exhibiting a distributionhalf-life of less than 1 minute and an elimination half-life ofapproximately 9 minutes.

The pharmacokinetic profiles of amifostine, active metabolite WR-1065and amifostine and WR-1065 combined following subcutaneousadministration of amifostine (500 mg dose) differ significantly fromthose obtained with intravenous administration. First, referring now toFIGS. 1 and 3, maximum whole blood concentrations are not reached by wayof an initial concentration spike or burst. Rather, blood concentrationsrise at a significantly slower rate, reaching a maximum approximately in5-60 minutes, preferably 10-40 minutes, after administration. Maximumlevels plateau, being maintained for about 10 to 130 minutes, preferablyabout 15 to 120 minutes, before gradually decreasing to baseline levels.

Additionally, the maximum whole blood concentrations of both amifostineand WR-1065 were significantly lower following subcutaneousadministration than intravenous administration. For example, forintravenous administration, peak levels for amifostine and WR-1065 wereabout 100 μM and about 23 μM, respectively. For subcutaneousadministration, maximum blood levels for amifostine and WR-1065 wereabout 12 μM and 4 μM, respectively. These concentrations arebiologically effective, both as a cytoprotective agent and in MDS (Dorret al., 1995, Eur. J. Cancer 31a (supp. 5):579, List et al., 1995, Blood86(10) Supp. 1:1327 (Abstract)).

Thus, the advantageous pharmacokinetic profiles of the invention aregenerally characterized by three main features:

(i) a first region wherein the plasma and/or whole blood concentrationof administered compound (and/or an active metabolite thereof) slowlyrises to a maximum level; (ii) a second region wherein the maximumplasma and/or whole blood concentration of administered compound (and/oran active metabolite thereof) plateaus; and (iii) a third region whereinthe maximum plasma and/or whole blood concentration of administeredcompound (and/or an active metabolite thereof) slowly decreases tobaseline levels.

In the first region of the pharmacokinetic profile, the plasma and/orblood concentration usually increases at a rate of about 0.1 μM/min. to40 μM/min., preferably about 0.3 μM/min. to 20 μM/min., and mostpreferably about 0.5 μM/min. to 10 μM/min. This increase toward themaximum concentration usually occurs over a period of about 1 min. to 60min., commonly over a period of about 5 min. to 25 min., typically overa period of about 10 min. to 20 min., and preferably over a period ofabout 15 min. The maximum plasma and/or blood concentration is usuallyreached in about 5 to 60 min. following administration, and ispreferably reached about 12 min. to 18 min. following administration.The increase in plasma and/or blood concentrations in this first regionis usually zero-order, i.e., the rate of increase is substantiallyconstant during the period of rise.

The maximum plasma and/or blood concentration of administered compound(and/or an active metabolite thereof) reached at the end of the firstregion remains relatively constant, i.e. plateaus, in the second regionof the pharmacokinetic profile. Preferably, the plasma and/or bloodconcentration does not fluctuate by more than about ±75%. Morepreferably, the plasma and/or blood concentration does not fluctuate bymore than ±35%. The plateau is usually maintained for about 10 min. to130 min., preferably about 15 min. to 120 min., and occurs about 15 min.to 80 min. after administration.

In the third region, the plasma and/or blood concentration ofadministered compound (and/or an active metabolite thereof) slowlydecreases towards baseline levels. The rate of decrease in plasma and/orblood concentration is typically governed by the subject's metabolism,and is not believed to be a critical feature towards effecting areduction or decrease in undesirable side-effects followingadministration. While the actual rate of decrease in the plasma and/orblood concentration of administered compound (or active metabolitesthereof) will vary from subject to subject, the concentration usuallydecreases at a rate of about 0.001 μM/min. to 0.2 μM/min., over a periodof about 30 min. to 220 min. The third region usually occurs about 60min. to 180 min. following administration.

While not intending to be bound by any particular theory, it is believedthat the reduction or decrease in adverse side-effects followingadministration is due to the characteristics of the above-describedpharmacokinetic profile. The slow rate at which the plasma and/or bloodconcentration of administered compound (and/or an active metabolitethereof) increases to maximum, as well as maintenance of the maximumlevel for a period of time, are thought to be of particular importance.Thus, it is believed that the pharmacokinetic profiles of the inventiondecrease or reduce adverse side effects of the compounds by eliminatingthe initial “spike” or “burst” in compound and/or metabolite plasmaand/or blood concentrations that are associated with conventionalintravenous administration, and that are likely unnecessary forefficacy.

The actual maximum plasma and/or blood concentration of administeredcompound (and/or an active metabolite thereof) is not believed to be ofcritical importance in reducing or decreasing adverse side-effects. Aslong as the compounds are administered according to the pharmacokineticprofiles described herein, a reduction or decrease in undesirableside-effects should be observed regardless of the actual maximum plasmaand/or blood concentration reached. Thus, as will be discussed in moredetail in a later section, virtually any amount of compound that yieldsa plasma and/or whole/blood concentration of the administered compound,and/or an active metabolite thereof, that is therapeutically effectivecan be advantageously administered according to the pharmacokineticprofiles described herein. The maximum plasma and/or blood concentrationwill usually range from about 1 μM to 40 μM.

5.3 Uses of the Methods

The methods of the invention can be used to efficaciously administer thecompounds described herein to patients to treat virtually any disorderthat is now known or that is later discovered to be treatable with suchcompounds.

For example, as the compounds described herein are able to selectivelyprotect normal tissues against the toxicities associated with ionizingradiation without adversely affecting the tumor response (Constine etal., 1986, “Protection by WR-2721 of Human Bone Marrow FunctionFollowing Irradiation” Int. J. Radia. Oncol. Biol. Phys. 12:1505-8; Liuet al., 1992, “Use of Radiation with or Without WR-2721 in AdvancedRectal Cancer” Cancer 69(11):2820-5; Wadler et al., 1993, “Pilot Trialof Cisplatin, Radiation and WR-2721 in Carcinoma of the Uterine Cervix:A New York Gynecologic Oncology Group Study” J. Clin. Oncol.11(8):1511-6; Buntzel et al., 1996, “Ethyol (Amifostine) ProvidesMultilineage heatoprotection and Protection Against NonhematologicToxicities Reduced by Radiochemotherapy (RCT) of Head and Neck Cancer,”Blood 88 (10) Supp. 1:448a [1781] [Abstract]), the methods describedherein can be used to administer the compounds to cancer patientsreceiving radiation therapy. Administering the compounds of theinvention to patients receiving radiation therapy can protect thepatients against the toxicities associated with the exposure toradiation therapy. These toxicities include, but are not limited to,neurotoxicity, nephrotoxicity, ototoxicity, cardiotoxicity, alopecia,mucositis, and include xerostomia, infertility, pulmonary toxicity, andrenal failure that can occur in severe cases. The compounds of theinvention are particularly effective in preventing mucositis andxerostomia. The compounds of the invention are also effective attreating acute xerostomia and late xerostomia and acute mucositis andlate mucositis. In particular the methods described herein can be usedto administer the compounds of the invention to patients receivingradiation therapy for head and neck cancer.

The term “xerostomia,” as used herein means dryness of mouth due tosalivary gland dysfunction induced by radiation. The term “acutexerostomia,” as used herein means xerostomia occurring within 90 days oftreatment initiation, characterized by dryness of mouth with thick,sticky saliva, altered taste, or acute salivary gland necrosis. The term“late xerostomia,” as used herein means xerostomia occurring 90 days to2 years after treatment initiation, characterized by dryness, poorsaliva production, or fibrosis of salivary glands.

The term “mucositis,” as used herein means inflammation of mucosalmembranes induced by radiation or chemotherapy. The term “acutemucositis,” as used herein means mucositis occurring within 90 days oftreatment initiation, characterized by parchy or confluentpseudomembranes, ulceration, or necrosis. The term “late mucositis,” asused herein means mucositis: occurring 90 days to 2 years aftertreatment initiation, characterized by mucosal atrophy, dryness,telangiectasia or ulceration.

The compounds described herein are also capable of selectivelyprotecting normal tissues from the toxicities associated with cancerchemotherapeutic agents, including but not limited to, alkylatingagents, platinum agents, anthracyclines and taxanes (Kemp et al., 1996,“Amifostine Pretreatment for Protection Against Cyclophosphamide-andCisplatin-Induced Toxicities: Results of A Randomized Control Trial inPatients with Advanced Ovarian Cancer” J. Clin. Oncol. 14:2101-12;Wasserman et al., 1981 “Differential Protection Against CytotoxicChemotherapeutic Effects on Bone Marrow CFUs by WR-2721 Cancer Clin.Trials 4:3-6; Glover et al., 1986, “WR-2721 Protects Against theHematologic Toxicity of Cyclophosphamide: A Controlled Phase II Trial”J. Clin. Oncol. 4:584-8; Schiller et al., 1996, “Amifostine, Cisplatinand Vinblastine in Metastic Nonsmall Cell Lung Cancer: A Report of HighResponse Rates and Prolonged Survival” J. Clin. Oncol. 14:1913-21; Dorret al., 1995, “Selective Cardioprotection of Rat Heart Myocytes Exposedto DNA Zutercalating Agents Using Amifostine (AMI) and It'sDephospharylated Metabolite, WR-1065,” Eur. J. Cancer 31a(Supp. 5):579;Betticher et al., 1995, “Carboplatin Combined with Amifostine, a BoneMarrow Protectant, in the Treatment of Non-Small Cell Lung Cancer: ARadomised Phase II Study” Br. J. Cancer 5:1551-5; DiPaola et al., 1996,“A Phase I Study of Amifostine and Paclitaxel in Patients with AdvancedMalignancies” Proc. Amer. Soc. Clin. Oncol. 15:488 (1556) Abstract).Thus, the methods of the invention can also be used to advantageouslyadminister the compounds described herein to cancer patients receivingchemotherapy.

The compounds described herein are also capable of stimulating bonemarrow growth (WO 96/25045) and causing the bone marrow function to morerapidly recover following chemotherapy (List et al., “AmifostineStimulated Formation of Multipotent Progenitor and Generated MacroscopicColonies in Normal and Myelodysplastic Bone Marrow,” Proc. Am. Soc.Clin. Oncol. 15:449 [1403] [Abstract]; List et al., 1996, “AmifostineProtects Primitive Hematopoietic Progenitors Against ChemotherapyCytotoxicity,” Semin. Oncol. 23 (4) Supp. 8:58-63). Thus, the methods ofthe invention further provide a useful means for administering thesecompounds to patients suffering from diseases requiring bone marrowgrowth, such as myelodysplastic syndrome (MDS), and to patients whosebone marrow has been exposed to chemotherapy. In addition, the methodsof the invention also provide a useful means for administering thecompounds to patients suffering from cancer and human immunodeficiencyvirus infection.

Administering aminoalkyl phosphorothioate and/or aminoalkyl thiolcompounds to patients according to the methods of the invention providesmyriad advantages over currently available intravenous modes ofadministration. A significant advantage is the reduction or decrease inundesirable side-effects suffered by patients receiving the therapy.Additionally, since the methods do not require i.v. injection, which isthe mode of administration most disliked by patients, the methodsdescribed herein will generally provide better patient compliance.Further, the methods of the invention do not necessarily requireadministration by skilled practitioners, making the therapy moreconvenient for patients.

5.4 Formulation and Routes of Administration

The compounds described herein, or pharmaceutically acceptable additionsalts or hydrates thereof, can be delivered to a patient so as to avoidor reduce undesirable side effects according to the invention using awide variety of routes or modes of administration. The only requirementis that the compound, and/or an active metabolite thereof, be deliveredaccording to the pharmacokinetic profiles described herein. Suitableroutes of administration include, but are not limited to, inhalation,transdermal, oral, rectal, transmucosal, intestinal and parenteraladministration, including intramuscular, subcutaneous and intravenousinjections.

For any mode of administration, the actual amount of compound delivered,as well as the dosing schedule necessary to achieve the advantageouspharmacokinetic profiles described herein, will be depend, in part, onsuch factors as the bioavailability of the compound (and/or an activemetabolite thereof), the disorder being treated, the desired therapeuticdose, and other factors that will be apparent to those of skill in theart. The actual amount delivered and dosing schedule can be readilydetermined by those of skill without undue experimentation by monitoringthe blood plasma levels of administered compound and/or an activemetabolite thereof, and adjusting the dosage or dosing schedule asnecessary to achieve the desired pharmacokinetic profile.

For example, for intravenous administration the advantageous profiles ofthe invention can be obtained by utilizing a significantly slower rateof infusion than is conventionally used, or by using an ambulatory pump.Methods of obtaining the desired pharmacokinetic profiles via othermodes of administration will be apparent to those of skill in the art,especially in light of the detailed disclosure provided herein.

The compounds described herein, or pharmaceutically acceptable saltsand/or hydrates thereof, may be administered singly, in combination withother compounds of the invention, and/or in combination with othertherapeutic agents, including cancer chemotherapeutic agents. The activecompound(s) may be administered alone or in the form of a pharmaceuticalcomposition, wherein the active compound(s) is in admixture with one ormore pharmaceutically acceptable carriers, excipients or diluents.Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP).

If desired, disintegrating agents may be added, such as the cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. It ispreferred that the compounds be administered by continuous infusionsubcutaneously over a period of 15 minutes to 24 hours. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

5.5 Effective Dosages

Pharmaceutical compositions suitable for use with the present inventioninclude compositions wherein the active ingredient is contained in atherapeutically effective amount, i.e., an amount effective to achieveits intended purpose. Of course, the actual amount of active ingredientwill depend on, among other things, its intended purpose. For example,when administered to cancer patients as a cytoprotectant in conjunctionwith radiation or chemotherapy, such compositions will contain an amountof active ingredient effective to, inter alia, ameliorate the harmfuleffects of ionizing radiation or chemotherapeutic agents to normaltissues. When administered to patients suffering from diseases requiringbone marrow growth, such as MDS, or more rapid recovery of bone marrowfunction following chemotherapy, such compositions will contain anamount of active ingredient effective to stimulate bone marrowproduction or function, prevent the development of or alleviate theexisting symptoms of, or prolong the survival of, the patient beingtreated. Determination of an effective amount is well within thecapabilities of those skilled in the art, especially in light of thedetailed disclosure herein.

For any compound described herein the therapeutically effective amountcan be initially estimated from cell culture assays. For example, a dosecan be formulated in animal models to achieve a circulatingconcentration range of compound, and/or an active metabolite thereof,that includes an effective concentration as determined in cell culture.Such information can be used to more accurately determine useful dosesin humans. See, e.g., Washburn et al., 1976, Prediction of the EffectiveRadioprotective Dose of WR-2721 in Humans Through an Interspecies TissueDistribution Study” Radiat. Res. 66:100-5.

Therapeutically effective amounts for use in humans can also beestimated from animal models. For example, a dose for humans can beformulated to achieve a circulating concentration found to be effectivein animals.

A therapeutically effective dose can also be estimated from humanpharmacokinetic data. While not intending to be bound by any particulartheory, it is believed that efficacy is related to a subject's totalexposure to an applied dose of administered drug, and/or an activemetabolite thereof, as determined by measuring the area under the bloodconcentration-time curve (AUC). Thus, a dose administered according tothe methods of the invention that has an AUC of administered compound(and/or an active metabolite thereof) within about 50% of the AUC of adose known to be effective for the indication being treated is expectedto be effective. A dose that has an AUC of administered compound (and/oran active metabolite thereof) within about 70%, 80% or even 90% or moreof the AUC of a known effective dose is preferred. Adjusting the dose toachieve maximal efficacy in humans based on the methods described above,particularly on the blood concentration and duration of administeredcompound and/or its active metabolites is well within the capabilitiesof the ordinarily skilled artisan.

For use as a cytoprotectant to selectively protect against thetoxicities of ionizing radiation or chemotherapeutic agents, acirculating concentration of administered compound (and/or and activemetabolite thereof) of about 2 μM to 100 μM is expected to be effective,with about 5 μM to 50 μM being preferred. Alternatively, or in addition,a tissue concentration of administered compound (and/or an activemetabolite thereof) of about 4 μM to 700 μM is expected to be effective,with about 20 μM to 350 μM being preferred.

Usual patient doses for administration of amifostine and/or its activemetabolite WR-1065 usually range from about 50 mg/day to 6000 mg/day,commonly from about 100 mg/day to 4000 mg/day, and typically from about200 mg/day to 3500 mg/day. Stated in terms of patient body weight, usualdosages range from about 0.6 to 100 mg/kg/day, commonly from about 1.1to 66 mg/kg/day, and typically from about 2.2 to 58 mg/kg/day. Stated interms of patient body surface areas, usual dosages range from about 23to 4000 mg/m²/day, commonly from about 45 to 2666 mg/m²/day, andtypically from about 90 to 2333 mg/m²/day.

For subcutaneous administration of amifostine and/or its activemetabolite WR-1065 patient dosages usually range from about 50 mg/day to1500 mg/day, commonly from about 100 mg/day to 1000 mg/day and typicallyfrom about 200 mg/day to 750 mg/day. Stated in terms of body weight,usual dosages range from 0.5 mg/kg/day to 25 mg/kg/day, commonly fromabout 1 mg/kg/day to 16 mg/kg/day and typically from about 3.3 mg/kg/dayto 12.5 mg/kg/day. Stated in terms of patient body surface areas, usualdoses range from about 22 mg/m²/day to 1000 mg/m²/day, commonly fromabout 45 mg/m²/day to 666 mg/m²/day and typically from about 133mg/m²/day to 500 mg/m²/day.

For use as a radioprotectant against the toxicities of ionizingradiation or as a chemoprotectant against the toxicities of cancertherapy, the dose should be administered enough in advance of exposureto radiation or chemotherapy to provide effect. For i.v. administration,the dose is preferably administered within 30 min. prior to theadministration of radiation or chemotherapy. For subcutaneousadministration, the dose is preferably administered within about 3 hoursprior to the administration of radiation therapy. More preferably thedose is subcutaneously administered about 20 to 90 minutes prior to theadministration of radiation therapy and most preferably about 1 hourprior to the administration of radiation therapy.

Administering the compounds of the invention are particularly useful intreating the toxicities associated ionizing radiation, especially inpatients with head and neck cancer. In these cases subcutaneousadministration is preferable and is most effective at reducing sideeffects.

For use in treating diseases requiring bone marrow growth, such as MDS,or recovery of bone marrow function, a circulating concentration ofadministered compound (and/or an active metabolite thereof) of about 2μM to 100 μM is expected to be effective. Alternatively, or in addition,a tissue concentration of administered compound (and/or an activemetabolite thereof) of about 0.1 μM to 1000 μM is expected to beeffective, with about 10 μM to 500 μM being preferred.

Usual patient doses for administration of amifostine and/or its activemetabolite WR-1065 usually range from about 50 mg/day to 1000 mg/day,commonly from about 100 mg/day to 900 mg/day, and typically from about200 mg/day to 800 mg/day. Stated in terms of patient body weight, usualdosages range from about 0.5 to 16 mg/kg/day, commonly from about 1.1 to15 mg/kg/day, and typically from about 2.2 to 13.5 mg/kg/day. Stated interms of patient body surface areas, usual dosages range from about 22to 666 mg/m²/day, commonly from about 45 to 600 mg/m²/day, and typicallyfrom about 90 to 540 mg/m²/day.

For subcutaneous administration of amifostine and/or its activemetabolite WR-1065 patient dosages usually range from about 50 mg/day to1200 mg/day, commonly from about 100 mg/day to 1100 mg/day and typicallyfrom about 200 mg/day to 1000 mg/day. Stated in terms of body weight,usual dosages range from 0.5 mg/kg/day to 20 mg/kg/day, commonly fromabout 1.1 mg/kg/day to 18 mg/kg/day and typically from about 2.2mg/kg/day to 16.2 mg/kg/day. Stated in terms of patient body surfaceareas, usual doses range from about 22 mg/m²/day to 800 mg/m²/day,commonly from about 45 mg/m²/day to 720 mg/m²/day and typically fromabout 90 mg/m²/day to 650 mg/m²/day.

For use as a radioprotectant against the toxicities of ionizingradiation, the dose is preferably at least about 500 mg, preferablyabout 500 mg to 1500 mg administered subcutaneously. Preferably, thedose is administered as two subcutaneous injections.

For other modes of administration, dosage amount and interval can beadjusted individually to provide effective plasma and/or tissue levelsof the administered compound, and/or an active metabolite thereof,according to the pharmacokinetic profiles described herein, aspreviously described.

The actual amount of composition administered will, of course, bedependent on the subject being treated, the subject's weight, theseverity of the affliction, the mode of administration and the judgementof the prescribing physician.

6. EXAMPLE Subcutaneous Administration of Amifostine Reduces In vivoToxicities

The advantageous effects of subcutaneously administering amifostine topatients was demonstrated in a Phase I, randomized three-way crossoverstudy. The study was conducted at one site in the United States.

Normal subjects were randomized to receive amifostine for threesuccessive days as an intravenous infusion (200 mg/m²), an oralformulation (500 mg) and a subcutaneous injection (500 mg) as describedin TABLE 1. A total of 12 subjects were treated in the study.

TABLE 1 Treatment Scheme (Three-Way Crossover Design) Day 1 Day 2 Day 3Sequence 1 A B C Sequence 2 B C A Sequence 3 C A B Treatment A:Amifostine (200 mg/m²) as a continuous intravenous infusion over 7.5minutes. Treatment B: Amifostine (500 mg) in liquid formulation as asingle oral dose. Treatment C: Amifostine (500 mg) as two simultaneoussubcutaneous injections.

All subjects were sequestered at the study site from the evening beforethe administration of the first dose of study drug until the final wholeblood sample was obtained. Whole blood samples were collected up to 4hours after each dose of amifostine.

Eligible subjects included healthy, normal male volunteers between theages of 18 and 35 years (inclusive). All subjects who entered the studyhad pretreatment values for complete blood counts (CBC), serumchemistries and urinalysis within ±10% of the normal range for thereferring laboratory which were considered clinically insignificant bythe inventors. Additionally, all subjects had a normal pretreatmentelectrocardiogram (EKG).

Subjects who were known to be human immunodeficiency virus (HIV)positive, active substance abusers or smokers (at least within the past6 months) were excluded. Also excluded were subjects with pretreatmenthypertension (systolic blood pressure >140 mm Hg), subjects with knowncardiovascular disease, subjects with general or psychologicalconditions which would preclude them from completing the study orsigning the informed consent, and subjects who were unwilling or unableto abstain from alcoholic beverages and all medications, includingprescription and over-the-counter drugs and vitamins, for 7 days priorto study entry and for 3 days while participating in the study.

6.1 Preparation of Intravenous Amifostine

For intravenous infusion, each vial of amifostine (500 mg per vial, U.S.Bioscience, lot. no. C3017C) was reconstituted with 9.7 mL of 0.9%Sodium Chloride Injection, USP. The appropriate dose of amifostine (200mg/m²) was further diluted with 0.9% Sodium Chloride Injection, USP, toproduce a volume of 50 mL for administration.

6.2 Preparation of Oral Amifostine

For oral administration, each vial of amifostine (500 mg per vial, U.S.Bioscience, lot. no. C3017C) was reconstituted with 5 mL of normalsaline solution. The reconstituted solution was drawn up in a 10 mLsyringe for administration.

6.3 Preparation of Subcutaneous Amifostine

For subcutaneous injection, each vial of amifostine (500 mg per vial,U.S. Bioscience, lot. no. C3017C) was reconstituted with 2.5 mL ofnormal saline solution. This solution was divided into two syringes(1.25 mL per syringe) for administration.

6.4 Administration of Intravenous Amifostine

Subjects received intravenous amifostine at a dose of 200 mg/m² as a 7.5minute infusion (via an infusion pump) beginning at 8:00 AM. During theinfusion, the subjects were kept in a supine position. Followingamifostine administration, all subjects were allowed to have a standardbreakfast. Moreover, all subjects were given a standard lunch at noon, astandard dinner at 6:00 PM and a standard snack at 10:00 PM.

Baseline systolic blood pressure and pulse were measured just prior tothe amifostine infusion, every 2.5 minutes during the infusion and 5minutes after the infusion. The amifostine infusion was interrupted ifsystolic blood pressure decreased as outlined in TABLE 2, or if thesubject developed symptoms related to decreased blood pressure(dizziness, diaphoresis or chest pain).

If hypotension occurred, the subject was to receive a rapid infusion ofnormal saline and was to be kept supine or in the Treudelenburg positionuntil the blood pressure returned to baseline. During this time, bloodpressure was monitored every 3 minutes.

If blood pressure returned to the value stipulated in TABLE 2 within 5minutes of stopping the amifostine infusion, and the subject wasotherwise asymptomatic, the amifostine infusion could be restarted withcontinued frequent monitoring of blood pressure. If any further episodeof hypotension occurred, the above guidelines were to be reapplied. If asubject's blood pressure did not return to the threshold value forrestarting the amifostine infusion within 5 minutes of stopping theamifostine infusion, the infusion was to be stopped and any unused drugwas discarded.

TABLE 2 Blood Pressure Guidelines for Interrupting and StartingAmifostine Baseline Systolic Blood Pressure (mm Hg) ≦110 111-130131-150 >170 Stop infusion if systolic ≦80 ≦100 ≦105 ≦130 blood pressuredecreases to: Re-start infusion when >80 >100 >120 >130 systolic bloodpressure returns to:

6.5 Administration of Oral Amifostine

Subjects receiving oral amifostine were to fast (except for water)beginning at midnight the night before receiving the oral dose untilnoon of the following day when they received a standard lunch. On theday subjects received oral amifostine, ranitidine (50 mg) wasadministered by intravenous injection 1 hour prior to the start ofamifostine administration.

Subjects received oral amifostine at a dose of 500 mg in a liquidformulation. This formulation was administered by gently squirting thesolution in the back of the subject's throat. Subjects were kept in asupine position for 1 hour following administration of the oral solutionof amifostine. Following administration of amifostine, all subjects wereallowed to drink 4 oz. of water and to eat hard candy to ameliorate anyunpleasant taste from oral amifostine. All subjects were given astandard lunch at noon, a standard dinner at 6:00 PM and a standardsnack at 10:00 PM.

Vital signs (blood pressure and pulse) were measured just prior to oraladministration of amifostine and repeated every 5 minutes for 1 hour. Ifhypotension occurred, the subject was to receive a rapid infusion of 500mL normal saline and was to be kept supine or in the Trendelenburgposition until blood pressure returned to baseline. During this time,blood pressure was monitored every 3 minutes.

6.6 Administration of Subcutaneous Amifostine

Subcutaneous amifostine was administered at a dose of 500 mg (as aliquid formulation). The dose of amifostine was divided equally into twosyringes and administered into two locations on the abdominal wall.Subjects were kept supine for 30 minutes following the two injections ofamifostine. Following amifostine administration, all subjects wereallowed to have a standard breakfast. All subjects were given a standardlunch at noon, a standard dinner at 6:00 PM and a standard snack at10:00 PM.

Vital signs (blood pressure and pulse) were measured just prior tosubcutaneous administration of amifostine and repeated every 5 minutesfor 30 minutes. If hypotension occurred, the subject was to receive arapid infusion of 500 mL normal saline and was to be kept supine or inthe Trendelenburg position until blood pressure returned to baseline.During this time, blood pressure was monitored every 3 minutes.

6.7 Prior and Concomitant Medications

Ranitidine was administered to all subjects on the day amifostine wasadministered orally. Any subject who experienced Grade 2 or highernausea and/or vomiting was allowed to receive prochlorperzaine (10 mgorally or by suppository) every 4 hours as needed.

6.8 Pretreatment Assessments

Pretreatment evaluations were performed within 7 days of initiatingtreatment and included the following:

history and physical exam including height, weight and vital signs(blood pressure, pulse and temperature);

CBC with differential and platelets;

blood chemistries including blood urea nitrogen (BUN), serum creatinine,calcium, total bilirubin, albumin, SGOT, SGPT, alkaline phosphate,glucose and total protein;

urinalysis; and

EKG.

If any subject's laboratory parameters were abnormal, the laboratorytests were repeated. If upon retest the parameters were normal, thesubject was included in the study. If the abnormal parameters persisted,the subject was excluded from the study.

6.9 Efficacy Assessments

The primary endpoint of this study was the relative bioavailability of500 mg of amifostine administered subcutaneous or orally compared to 200mg/m² of amifostine administered intravenously. As this was a crossoverstudy in which each subject received amifostine by all three routes ofadministration, the bioavailabilities of subcutaneous and oralamifostine relative to intravenous amifostine were assessed within eachsubject.

6.9.1 Collection of Blood Samples

Blood samples (5 to 7 mL each) were collected either by venipuncture orindwelling venous catheter (IVC) heparin lock. If IVC was used, it wasinserted in the arm opposite the infusion site. If central line wasused, intravenous amifostine was administered by a distal intravenoussite. For either line, the first 3 to 5 cc of blood were discarded (voidvolume) prior to blood collection.

For subjects receiving intravenous amifostine, blood samples werecollected 5 minutes prior to amifostine administration (baseline) and2.5, 5, 7.5, 10, 15, 20, 30 and 60 minutes, and 2 and 4 hours afteramifostine administration.

For subjects receiving oral amifostine as a liquid formulation, bloodsamples were collected immediately prior to amifostine administration(baseline) and 5, 10, 15, 30, 45 and 60 minutes, and 2 and 4 hours afteramifostine administration.

For subjects receiving subcutaneous amifostine, blood samples werecollected immediately prior to amifostine administration (baseline) and5, 10, 15, 30, 45 and 60 minutes, and 2 and 4 hours after amifostineadministration.

6.9.2 Blood Sample Preparation

Blood samples were collected at specified times during a 4-hour periodafter each dose of amifostine. Each blood sample was prepared asdescribed in TABLE 3. Blood samples were divided for purposes ofanalysis: one sample was used to determine the presence of amifostine,and the other sample was used to determine the presence of WR-1065(active metabolite).

TABLE 3 Preparation of Plasma Samples for Determination ofConcentrations of Amifostine and WR-1065 Whole blood was drawn into EDTAvacutainer tubes on ice ↓ ↓ Removed 1.5 mL whole blood Remainder ofwhole blood and pipetted into after removing 1.5 mL polypropylene tubescontaining 1.5 mL PCA/EDTA solution on ice ↓ ↓ Immediately mixedCentrifuged tubes at high thoroughly by vortexing speed for 5 minutes at4° C. ↓ ↓ Centrifuged tubes at high Plasma supernatant removed speed for10 minutes at 4° C. into labelled polypropylene tubes on ice ↓ ↓ SaveClear supernatant removed Store at −70° C. (these Pellet into labelledpolypropylene samples are for tubes on ice determination of amifostine)↓ ↓ Store at Store at −70° C. (these −70° C. samples are fordetermination of WR-1065, the active metabolite) EDTA isethylenediaminetetraacetic acid PCA is perchloric acid

As shown in TABLE 3, the preparation of amifostine (parent drug) andWR-1065 (active metabolite) for pharmacokinetic analysis differed fromone another. This difference was based on the metabolic pathway ofamifostine. Amifostine is a prodrug that is dephosphorylated at thetissue site by alkaline phosphatase to the active metabolite, WR-1065.WR-1065 can be further oxidized to form both symmetrical and mixeddisulfides, or undergo further metabolism via copper-dependent amineoxidase to form acrolein and cysteamine. To prevent further oxidation ofWR-1065, PCA/EDTA was added to the samples prior to centrifugation.Because PCA/EDTA ruptures the cellular content of the blood, the sampleswere centrifuged for a longer period of time to preventcontamination/metabolism of the active metabolite. Consequently, onlythe clear supernatant (serum) was used for analysis of WR-1065. Incontrast, no PCA/EDTA was added to the blood samples designated foranalysis of the parent drug; therefore, plasma samples were prepared foranalysis of the parent drug.

6.9.3 Pharmacokinetic Analysis

Concentrations of amifostine (unchanged drug) and WR-1065 (activemetabolite) were measured using electrochemical-detection high-pressureliquid chromatography (HPLC) methods as described by Shaw, et al. (Shawet al. 1984, “A Liquid Chromatographic Electrochemical Assay forS-2-(3-Aminopropylamino) ethylphosphorothioate (WR-2721) in HumanPlasma,” J. Liq. Chromatog. 7:2447-2465; Shaw et al., 1986, “Measurementof S-2-(3-Aminopropyl Amino) ethanethiol (WR-1065) in Blood and Tissue,”J. Liq. Chromatog. 9:845-859). WINNONLIN, the WINDOWS version ofPCNONLIN, was used for the determination of pharmacokinetic parametersfor each subject following each route of administration.Non-compartmental modelling of the data was performed.

6.10 Safety Assessments

All subjects who had received at least one dose of protocol therapy wereconsidered evaluable for safety. Safety was evaluated during and afteradministration of amifostine (prior to subjects leaving the study site)via analyses of adverse events, physical examination and laboratorytests. Toxicity notation was done after each dose of amifostine.Toxicity was based on National Cancer Institute (NCI) Common ToxicityCriteria.

The following safety evaluations were done after the last dose ofamifostine (on Day 3) but prior to subjects leaving the study site:

physical exam including vital signs (blood pressure, pulse andtemperature);

CBC with differential and platelets;

blood chemistries including BUN, serum creatinine, calcium, totalbilirubin, albumin, SGOT, SGPT, alkaline phosphatase, glucose and totalprotein;

urinalysis; and

toxicity notation.

Subjects who had abnormal laboratory values at discharge had thoseparameters repeated. If the parameters remained abnormal, the subjectwas referred to a physician of his choice for follow-up.

6.11 Sample Size Determination

Using a 3×3 latin square method with 12 subjects, the power to detect a30% difference in area under the curve (AUC) for amifostine (parentdrug) and WR-1065 was 80%. This difference was defined as a change inAUC values (67% more or 40% less) for one route of administration versusthe other two routes of administration. The power calculation assumed astandard deviation of 50% of the mean AUC and a 70% correction withsubjects.

6.12 Statistical Analysis

The primary endpoint of this study was the relative bioavailability of500 mg of amifostine administered orally or subcutaneously compared to200 mg/m² of amifostine administered intravenously. As this was acrossover study in which each subject received amifostine by all threeroutes of administration, the bioavailabilities of subcutaneous and oralamifostine relative to intravenous amifostine were assessed within eachsubject.

Bioavailability was assessed using area under the plasma/serumconcentration-time curve (AUC) from 0 to 4 hours. AUCs were calculatedusing extrapolation according to a straightline from the last non-zeropoint.

The bioavailability of amifostine (parent drug plus active metabolite)following oral and subcutaneous administration was based on the ratio ofAUCs of these routes of administration with the AUC of intravenousamifostine. The AUCs were then analyzed using an analysis of variance(ANOVA) model with route, period and subject as variables. Sequence wasnot included in the model as this variable was uniquely determined byroute and period. The confidence interval on the ratio of thesubcutaneous or oral route to the intravenous route was calculated usingthe mean square error from the entire ANOVA model.

Because this was a crossover study to be performed on three successivedays, toxicities were assigned to the route of administration on the daythe toxicity occurred. A full nonparametric crossover model (likelihoodof ratio test) was used to assess toxicities which corrected for periodand sequence effects. A PROC CATMOD of SAS@ was used for this analysis.

6.13 Results

Between Apr. 2, 1996 and Jul. 23, 1996, 12 healthy volunteers wereenrolled onto this Phase I bioavailability study of amifostine givenintravenously, orally and subcutaneously (Protocol WR-A057). This studywas conducted at one site in the United States. All 12 subjectscompleted the study as per the protocol.

A total of 12 healthy men enrolled onto Protocol WR-A057 (TABLE 4). Themedian age of these subjects was 25.5 years, ranging from 18 to 34years. The median body surface area (BSA) was 1.84 m², ranging from 1.69to 2.15 m². All subjects had normal pretreatment laboratory valueswithin 10% of the normal range and no vital sign abnormalities prior toreceiving study medication. Moreover, all subjects had normalpretreatment EKG.

TABLE 4 Baseline Demographic Characteristics of the 12 Subjects WhoReceived Amifostine Intravenously, Orally and Subcutaneously NumberParameter (n = 12) Percent Age (years) Median 25.5 Range 18-34 RaceCaucasian 6  (50.0%) Black 5  (41.7%) Other 1   (8.3%) Body Surface Area(m²) Median  1.84 Range 1.69-2.15 Weight (kg) Median 70.2 Range 59-91Height (cm) Median 176.5  Range 158-188

6.13.1 Bioavailability

FIG. 1 shows the plasma/serum concentration-time curves for amifostine(parent drug plus active metabolite) following oral, subcutaneous andintravenous administration of amifostine in all 12 subjects. As seen inthis figure, the shape of the plasma/serum concentration-time curves ofthe three routes of administration were markedly different. Following a200 mg/m² intravenous infusion (over 7.5 minutes), there was rapiduptake of amifostine within <1 minute of drug administration.Thereafter, plasma/serum concentrations of amifostine decreased rapidly.The maximum concentration of amifostine was approximately 130 μM 8 to 10minutes after intravenous administration which dropped below 10 μM 45minutes after drug administration.

Following a 500 mg subcutaneous injection, there was a markedly slowerrise in plasma/serum concentrations of amifostine as compared tointravenous administration. The maximum concentration of amifostine(approximately 15 μM) was observed 15 minutes after subcutaneousadministration and was maintained for approximately 30 minutes.Thereafter, concentrations of amifostine fell below 10 μM approximately50 minutes after subcutaneous administration. Thirty minutes followingadministration of 200 mg/m² intravenous amifostine and 500 mgsubcutaneous amifostine, the plasma/serum concentration-time curves ofamifostine (parent drug plus active metabolite) were identical.

Following oral administration (500 mg as an liquid formulation), themaximum concentration of amifostine (approximately 5 μM) was observed 5to 8 minutes after drug administration. Concentrations of amifostinedropped below 1.0 μM at 10 to 13 minutes after oral drug administration.A second peak of approximately 4 μM was observed 45 minutes after oraldrug administration which dropped to approximately 1 μM at 60 minutesafter drug administration.

FIG. 2 shows the average plasma concentration-time curves for amifostine(parent drug) following oral, subcutaneous and intravenousadministration of amifostine to the 12 subjects. There was no detectableamount of parent drug in plasma samples of subjects following oralamifostine. The shape of the plasma concentration-time curves of theintravenous and subcutaneous dose were similar to those seen in FIG. 1.Following the 200 mg/m² intravenous dose, there was rapid uptake ofamifostine within <1 minute of drug administration. Thereafter, plasmaconcentrations of amifostine decreased rapidly. The maximumconcentration of amifostine was approximately 100 μM 8 to 10 minutesafter intravenous administration which dropped below 10 μM approximately30 minutes after drug administration. Following a 500 mg subcutaneousinjection, there was a slower rise in plasma concentrations ofamifostine. The maximum concentration of amifostine (approximately 10μM) was observed 15 minutes after subcutaneous and was maintained forapproximately 30 minutes. Thereafter, concentrations of amifostine fellbelow 10 μM approximately 45 minutes after subcutaneous administration.Thirty minutes following administration of 200 mg/m² intravenousamifostine and 500 mg subcutaneous amifostine, the plasmaconcentration-time curves of the parent drug were similar but withhigher concentrations of parent drug noted for subcutaneous amifostineversus intravenous amifostine.

FIG. 3 shows the average serum concentration-time curves for WR-1065(active metabolite) following subcutaneous, oral and intravenousadministration of amifostine in all 12 subjects. As seen in this figure,the shape of the serum concentration-time curves of the three routes ofadministration were similar to those observed in FIG. 1. In fact, theserum concentration-time curve for oral amifostine in FIG. 1 was theexact same curve as in FIG. 3; this is due to the fact that there was nodetectable amount of parent drug in the plasma samples of subjectsfollowing oral amifostine. Following a 200 mg/m² intravenous infusion,there was rapid uptake of WR-1065 within <1 minute of drugadministration. Thereafter, serum concentrations of WR-1065 alsodecreased rapidly but at a slower rate than that which was observed withthe parent drug (see FIG. 2). The maximum concentration of WR-1065 wasapproximately 30 μM 10 minutes after intravenous administration whichfell below 5 μM approximately 45 minutes after drug administration.Following a 500 mg subcutaneous injection, there was a slower rise inserum concentrations of WR-1065. The maximum concentration of WR-1065(approximately 5 μM) was observed 15 minutes after subcutaneousadministration and was maintained for approximately 30 minutes.Thereafter, concentrations of WR-1065 fell below 5 μM approximately 60minutes after subcutaneous administration. Forty minutes followingadministration of 200 mg/m² intravenous amifostine and 500 mgsubcutaneous amifostine, the serum concentration-time curves of theactive metabolite were identical.

The primary endpoint of this study was the relative bioavailability of500 mg amifostine administered orally or subcutaneously to 200 mg/m²amifostine administered intravenously. TABLE 5 lists the AUC values ofamifostine (parent drug and active metabolite combined) for each subjectfollowing oral, subcutaneous and intravenous administration ofamifostine. Based on the ratio of AUCs, the relative bioavailability ofamifostine (parent drug and active metabolite) following a 500 mgsubcutaneous dose was 0.67 (95% confidence interval of 0.37 to 0.98) ofthe 200 mg/m² intravenous dose. The relative bioavailability ofamifostine following a 500 mg oral dose was 0.11 (95% confidenceinterval of 0.04 to 0.18) of the 200 mg/m² intravenous dose.

TABLE 5 Relative Bioavailability of 500 mg Amifostine AdministeredOrally or Subcutaneously to 200 mg/m² Amifostine AdministeredIntravenously in 12 Healthy, Male Subjects (Parent Drug and ActiveMetabolite Combined) AUC (μM × min) of Combined 500 mg Sub- 500 mg 200mg/m² Bioavailability Subject cutaneous Oral Intravenous Ratio RatioNumber (SC) (PO) (IV) PO/IV SC/IV 1001 1726  656 1573 0.42 1.10 10022411  243 2820 0.09 0.86 1003 3946  456 2432 0.19 1.62 1004 475 131 21980.06 0.22 1005 643 378 1383 0.27 0.46 1006 180 132 1876 0.07 0.10 1007732  50 2102 0.02 0.35 1008 3080   42 1811 0.02 1.70 1009 550  86 12960.07 0.42 1010 348  0 1343 0.00 0.26 1011 985 121 1382 0.09 0.71 1012550  2 1832 0.00 0.30 Mean 1302  191 1837 0.11 0.67 95% CI* 611-199375-307 1565-2109 0.04-0.18 0.37-0.98 CI: confidence interval. *Using themean square error from the ANOVA model.

TABLE 6 lists the AUC values of the parent drug for each subjectfollowing oral, subcutaneous and intravenous administration ofamifostine. Based on the ratio of AUCs, the relative bioavailability ofthe parent drug following a 500 mg subcutaneous dose was 0.72 (95%confidence interval of 0.26 to 1.18) of the 200 mg/m² intravenous dose.The relative bioavailability of the parent drug following a 500 mg oraldose was 0.00 as no parent drug was detected in blood samples ofsubjects following oral administration of amifostine (see FIG. 2).

TABLE 6 Relative Bicoavailability of 500 mg Amifostine AdministeredOrally or Subcutaneously to 200 mg/m² Amifostine AdministeredIntravenously in 12 Healthy, Male Subjects (Parent Drug) AUC (μM × min)of Parent Drug 500 mg Sub- 500 mg 200 mg/m² Bioavailability Subjectcutaneous Oral Intravenous Ratio Ratio Number (SC) (PO) (IV) PO/IV SC/IV1001 1208 0  840 0.00 1.44 1002 1135 0 1305 0.00 0.87 1003 2883 0 12380.00 2.33 1004  224 0 1759 0.00 0.13 1005  305 0  805 0.00 0.38 1006   00 1526 0.00 0.00 1007  515 0 1723 0.00 0.30 1008 2924 0 1378 0.00 2.121009  154 0  812 0.00 0.19 1010  264 0  928 0.00 0.28 1011  697 0 11560.00 0.60 1012   0 0 1351 0.00 0.00 Mean  859 0 1235 0.00 0.72 95% CI*274-1444 — 1044-1426 — 0.26-1.18 CI: confidence interval. *Using themean square error from the ANOVA model.

TABLE 7 lists the AUC values of the active metabolite (WR-1065) for eachsubject following oral, subcutaneous and intravenous administration ofamifostine. Based on the ratio of AUCs, the relative bioavailability ofthe active metabolite following a 500 mg subcutaneous dose was 0.71 (95%confidence interval of 0.55 to 0.86) of the 200 mg/M² intravenous dose.The relative bioavailability of the active metabolite following a 500 mgoral dose was 0.32 (95% confidence interval of 0.16 to 0.48) of the 200mg/m² intravenous dose.

TABLE 7 Relative Bioavailability of 500 mg Amifostine AdministeredOrally or Subcutaneously to 200 mg/m² Amifostine AdministeredIntravenously in 12 Healthy, Male Subjects (Active Metabolite) AUC (μM ×min) of Active Metabolite 500 mg Sub- 500 mg 200 mg/m² BioavailabilitySubject cutaneous Oral Intravenous Ratio Ratio Number (SC) (PO) (IV)PO/IV SC/IV 1001 518 656 745 0.88 0.70 1002 1587  243 1583  0.15 1.001003 1047  456 1234  0.37 0.85 1004 251 131 424 0.31 0.59 1005 338 378569 0.66 0.59 1006 180 132 265 0.50 0.68 1007 218  50 381 0.13 0.57 1008142  42 437 0.10 0.32 1009 379  86 482 0.18 0.79 1010  84  0 338 0.000.25 1011 188 121 203 0.59 0.93 1012 550  2 457 0.00 1.20 Mean 457 191593 0.32 0.71 95% CI* 207-707 75-307 360-826 0.16-0.48 0.55-0.86 CI:confidence interval. *Using the mean square error from the ANOVA model.

The aforementioned AUC data presented in TABLES 5, 6 and 7 were used tocalculate the absolute bioavailability of amifostine following oral andsubcutaneous administration. Using the 200 mg/m² intravenous dose as thedose representing 100% bioavailability, the AUC data for oral andsubcutaneous amifostine were adjusted for dose and compared with the AUCdata for intravenous amifostine. Based on the ratio of AUCs, in contrastto the relative bioavailabilities, the absolute bioavailability ofamifostine (parent drug plus active metabolite) following subcutaneousadministration was 0.50 (95% confidence interval of 0.27 to 0.73) of theintravenous dose. The absolute bioavailabilities of the parent drug andthe active metabolite following subcutaneous administration were 0.53(95% confidence interval of 0.18 to 0.88) and 0.53, respectively, (95%confidence interval of 0.41 to 0.64) of the intravenous dose. Theabsolute bioavailability of amifostine (parent drug plus activemetabolite) following oral administration was 0.08 (95% confidenceinterval of 0.03 to 0.13) of the intravenous dose.

6.13.2 Efficacy

Plasma concentrations of amifostine (parent drug) and serumconcentrations of WR-1065 (active metabolite) were measured in bloodsamples obtained from 12 heathy, male subjects prior to and up to 4hours after receiving amifostine as a 500 mg subcutaneous injection, a500 mg oral solution and a 200 mg/m² intravenous infusion (over 7.5minutes). From these data, plasma/serum concentration-time curves weregenerated and used to determine the pharmacokinetic profile ofamifostine (both parent drug and active metabolite) following each routeof administration.

As shown in FIG. 1, the shapes of the plasma/serum concentration-timecurves for amifostine (parent drug plus active metabolite) of the threeroutes of administration were different. Following all three routes ofadministration, there was rapid uptake of amifostine within 5 minutes ofdrug administration. The intravenous route exhibited the fastest rate ofuptake, followed by the subcutaneous route and the oral route.Thereafter, plasma/serum concentrations of amifostine declined. The rateof decline was fastest for the intravenous route and slowest for thesubcutaneous route. The maximum concentration of amifostine (parent drugplus active metabolite) following intravenous administration wasapproximately 130 μM (approximately 100 μM for parent drug and 30 μM foractive metabolite). Maximum concentrations of amifostine (parent plusactive metabolite) after subcutaneous and oral administration wereapproximately 15 μM and 5 μM, respectively. Maximum concentrations werereached at approximately 15 minutes and plateaued until approximately 45minutes of drug administration. As shown in FIG. 2, there was nodetectable amount of parent drug in plasma samples of subjects followingoral amifostine.

6.13.3 Adverse Events

TABLE 8 lists those subjects who reported an adverse event followingintravenous, oral and subcutaneous administration of amifostine. Nosubject discontinued amifostine due to adverse events. All adverseevents were mild in severity, transient and considered related toamifostine. Five subjects reported adverse events following intravenousamifostine versus one subject each following oral and subcutaneousamifostine. Nausea was the most common adverse event. Other adverseevents included headache, hypotension, vomiting, lightheadedness andsomnolence.

TABLE 8 List of Subjects Who Reported Adverse Events* FollowingIntravenous, Oral and Subcutaneous Administration of Amifostine (N = 12)Adverse Event Intravenous Oral Subcutaneous Headache 1008 Hypotension1003 1003 Nausea 1001, 1003, 1002 1005, 1007, 1008 Vomiting 1008Lightheadedness 1003 1003 Somnolence 1008 *All adverse events were mildin severity and related to amifostine.

6.13.3.1 Hypotension

Mild and transient episodes of hypotension were reported by one subjectafter intravenous and oral administration of amifostine (TABLE 10). OnDay 2 of the study, Subject 1003 had a pre-infusion blood pressurereading of 115/58 mm Hg which decreased to 99/56 mm Hg, 5 minutes aftercompletion of the infusion. Five minutes later, his blood pressurereturned to baseline (114/56 mm Hg). Subject 1003 also experienced twoepisodes of hypotension following oral administration of amifostine onDay 3. With a baseline blood pressure reading of 121/70 mm Hg, transientdecreases in blood pressure (99/49 and 98/51 mm Hg) were observed at 5and 25 minutes after oral administration of amifostine, respectively.Thereafter, none of the other blood pressure readings met the criteriafor hypotension.

There was no incidence of hypotension following subcutaneousadministration of amifostine.

6.13.3.2 Emesis

Six subjects reported nausea and one subject vomited during the study.Nausea was reported in five subjects following intravenous amifostineand in one subject following subcutaneous amifostine (TABLE 10).Vomiting was reported in one subject following intravenous amifostine.All episodes of nausea/vomiting were mild in severity and transient innature. Antiemetic therapy was prescribed in two subjects followingintravenous amifostine. Subject 1007 received prochlorperazine (10 mg)for nausea, and Subject 1008 received prochlorperazine (10 mg) fornausea/vomiting.

6.13.3.3 Clinical Evaluations

Physical examinations including weight and vital signs (blood pressure,pulse and temperature) were performed after the last dose of amifostinebut prior to subjects leaving the study site. No remarkable changes werenoted in these parameters following the last dose of amifostine.

6.13.3.4 Clinical Laboratory Tests

Laboratory tests including hematology, serum chemistry and urinalysiswere performed after the last dose of amifostine but prior to subjectsleaving the study site. Laboratory results were compared to laboratoryreference ranges as well as to NCI Common Toxicity Criteria. One subject(Subject 1010) had a serum creatinine value (0.60 mg/dL) >10% below thelower limit of the reference range (0.70 to 1.40 mg/dL); his baselinevalue was 0.90 mg/dL. This low value was considered by the inventors asclinically insignificant. In addition, none of the subjects experienceda Grade 2 or higher laboratory toxicity according to NCI Common ToxicityCriteria.

6.13.3.5 Safety Conclusions

Amifostine was well tolerated regardless of route of administration. All12 subjects completed the required study medication as per the protocol.No subject discontinued amifostine due to adverse events. All adverseevents were mild in severity, transient and considered related toamifostine. Five subjects reported adverse events following intravenousamifostine versus one subject each following oral and subcutaneousamifostine (TABLE 8). Nausea was the most common adverse event. Otheradverse events included headache, hypotension, vomiting, lightheadednessand somnolence. Mild and transient episodes of hypotension were reportedby one subject after intravenous and oral administration of amifostine.These episodes occurred after amifostine administration, and none lastedmore than 5 minutes in duration. There was no incidence of hypotensionfollowing subcutaneous administration of amifostine. There were noremarkable findings for any of the clinical evaluations or clinicallaboratory tests.

7. EXAMPLE Subcutaneous Administration of Amifostine Protected AnimalsAgainst Radiation-induced Mucositis

One of the major limiting acute toxicities associated with radiotherapyis radiation-induced mucositis. The ability to reduce the duration andseverity of acute mucosal reactions is of particular importance in theradiotherapy and/or chemotherapy of head and neck cancer. Therefore, theradioprotective effects of amifostine were examined in an experimentalmodel of mucositis. In particular, the study compared theradioprotective effects of amifostine by subcutaneous (s.c.) andintraperitoneal (i.p.) administration. The mouse model developed byParkins et al. was used to examine the mucosal reactions in the inferiorlip of mice after irradiation, and this model has been established as areproducible model in the art (Parkins et al., 1983, Radiother. Oncol.1:159-165).

7.1 Experimental Design

C57BL/6 female mice of 8-10 weeks old were used and fed with semi liquidfood. A total of 40 mice were randomly divided into eight treatmentgroups of five mice each. The treatment groups were:

Group 1: Saline solution (i.p.) and irradiation

Group 2: Saline solution (s.c.) and irradiation

Group 3: Amifostine (200 mg/Kg, i.p.) and irradiation

Group 4: Amifostine (400 mg/Kg, is.) and irradiation

Group 5: Amifostine (200 mg/Kg, s.c.) and irradiation

Group 6: Amifostine (400 mg/Kg, s.c.) and irradiation

Group 7: Saline solution (i.p.)

Group 8: Saline solution (s.c.)

Unanesthetized mice were maintained in supine position and irradiatedexclusively on the tip of their mouth. They were immobilized using jigscomparable to those previously used by Ang et al. (1982, Int. J. Radiat.Oncol. Biol. Phys. 8:145-148). Irradiation was performed with a RT 250Philips apparatus delivering 1.98 Gy per min. (200 Kv, 20 mA, filter of0.2 mm de Cu). During irradiation, a constant normobaric air renewal wasmaintained. The effects of amifostine were evaluated using a single doseof 16.5 Gy.

7.2 Administration of Amifostine

Amifostine was dissolved in physiological saline (0.9% Nacl to achieve afinal concentration of 50 mg/ml) immediately before injections at 200 or400 mg/Kg body weight. Both i.p. and s.c. injections were conducted 30minutes before irradiation. A placebo solution of saline alone was usedfor the control group.

7.3 Mucositis Scoring System

The effects of irradiation on lip mucosa were evaluated using thescoring system described by Parkins et al. (1983, Radiother. Oncol.1:159-165). Body weight of the treated mice were scored daily aftertreatment. Reduction in body weight was used as an objective indicationof the severity of mucositis induced by irradiation, presumablyresulting from the inability of the animals to eat. In this model, theacute reactions peaked around day 10 to 11 after irradiation.

Other symptoms of mucositis such as mucosal erythema and edema were alsorecorded. These symptoms developed more slowly than weight lossfollowing irradiation. Mucosal erythema and edema were scoredseparately, and could be analyzed as separate scores or as a combinedscore yielding a maximum score of 7. Mouse lip mucosal erythema wasscored according to Table 9.

TABLE 9 Scoring System for Mucosal Erythema Score Mucosal Observation  0.5 doubtful if abnormally pink 1 slight but definitely reddening 2severe reddening 3 focal desquamation 4 exudation or crusting coveringabout ½ lip area 5 exudation or crusting covering more than ½ lip area

Mucosal edema (swelling) of the lips was scored according to Table 10.

TABLE 10 Scoring System for Mucosal Edema Score Mucosal Observation  0.5 50—50 doubtful if any swelling 1 slight but definitely swelling 2sever swelling

7.4 Results

Body weight reduction of irradiated mice was measured as an objectiveindicator of mucositis (Table 11). A single dose of irradiation greatlyreduced the body weight of the animals, particularly 7-13 days aftertreatment (FIGS. 4A and 4B). Non-irradiated animals maintained steadybody weight throughout the course of the study. Reduction in body weightwas prevented in animals which received either i.p. or s.c. injection ofamifostine. The radioprotective effects of amifostine were dosedependent.

Non-irradiated mice had no erythema during the entire period of theexperiment (Table 12). In contrast, erythema was observed in allirradiation groups (FIGS. 5A and 5B). Both i.p and s.c. administrationof amifostine decreased the average erythema score as compared to salineplus irradiation. Similarly, mucosal edema scores were decreased by bothinjection methods of amifostine (FIGS. 6A and 6B, Table 13).

The data presented in FIGS. 4A, 4B, 5A, 5B, 6A, 6B and Tables 11-13 showthat both s.c. and i.p. administration were effective in reducing theadverse effects of radiation, as measured by three mucositis indicators,i.e., body weight, erythema score and edema score. Furthermore, thesubcutaneous administration of amifostine at 200 mg/kg produced thehighest efficacy at the time points of maximal adverse effects (i.e.,day 9 for body weight and day 12 for erythema and edema). An increase inthe dose of amifostine from 200 mg/kg to 400 mg/kg produced minimaladditional benefits. Thus, these results indicate that subcutaneouslyadministered amifostine may have improved efficacy over other routes ofadministration, such as i.p.

TABLE 11 Body Weight 0 6 7 9 12 13 14 Saline (l.p.) + Irradiation Days 120 19.8 16.3 16.5 15.1 15.5 18.4 2 20 17.4 14.6 13.3 14.3 13.87 15.4 320 17.8 16 13.9 14.8 15.6 16.9 4 19.5 16.8 15.6 13.7 15.2 16.4 17.9 5 1918.2 16.1 15.4 15.1 14 16.8 Mean 19.7 18 15.72 14.56 14.9 15.074 17.08Standard 0.2 0.505964 0.302324 0.601332 0.164317 0.490893 0.517107Saline (s.c.) + Irradiation Mouse 1 20 20.1 18.8 18 17.6 18.46 20.9 2 2017.8 15.5 14.5 15.7 16 18.2 3 20 19 18.1 17.1 16.9 18.5 20.2 4 21 1917.7 16.4 17.7 19 21.5 5 20 18.2 15.7 14.4 16.6 17.5 18.8 Mean 20.218.82 17.16 16.08 16.9 17.892 19.92 Standard 0.2 0.395474 0.6615130.71232 0.364692 0.531662 0.622415 Amifostine (l.p.) at 200 mg/kg +Irradiation Mouse 1 20 18.1 19.1 18 19.3 19.3 19.8 2 20 18.1 16.7 1719.7 19.6 20.1 3 20 18.6 18.3 17.3 19.3 19.4 20 4 20 19 19.6 20.1 21.321.4 21.6 5 20 20 20.7 20.4 21.3 21 21 Mean 20 18.76 18.88 18.56 20.1820.14 20.5 Standard 0 0.352987 0.669627 0.710352 0.463033 0.44 0.343511Amifostine (l.p.) at 400 mg/kg + Irradiation Mouse 1 20.5 19.7 20 20.421.2 20.8 21.6 2 19 18.1 19.4 18.2 18.7 19 18.8 3 21 19.2 20.7 21.8 2121.3 21.8 4 19 19.4 20.4 20.8 20.8 21.2 21.1 Mean 19.875 19.1 20.12520.3 20.425 20.575 20.825 Standard 0.515388 0.348807 0.280995 0.7593860.580768 0.535996 0.690863 Amifostine (s.c.) at 200 mg/kg + IrradiationMouse 1 20 17.7 18.3 19.4 18.6 19.2 19.8 2 20 18.8 19.9 20.6 19.5 19.820.9 3 19.5 18.2 19.3 19.6 19.5 19.7 20.2 4 19 18.7 19 19.5 20.3 20.322.2 5 19 18.9 18.8 19.8 19.9 20 21.5 Mean 19.5 18.46 19.06 19.78 19.5619.8 20.92 Standard 0.223607 0.224944 0.265707 0.215407 0.2821350.181659 0.432897 Amifostine (s.c.) at 400 mg/kg + Irradiation Mouse 120 19 19.4 20.6 19.9 20.5 21.4 2 20 19.7 19.8 20.8 18.8 20.3 21 3 19.520 20.6 21.4 20.7 21.1 21.4 4 21 16.9 19.2 20.7 21.7 22 24 5 20 19.6 2020.7 20.8 21.3 21.7 Mean 20.1 19.04 19.8 20.84 20.38 21.04 21.9 Standard0.244949 0.559106 0.244949 0.143527 0.487237 0.302655 0.536656 Saline(l.p.) Mouse 1 20 20.7 19.8 20.4 19.9 20.7 20.9 2 19.3 19.6 19.1 20.520.1 19.9 20.5 3 20.6 21.1 21.1 21.5 21.5 21.5 23.1 4 19.3 20.2 20.120.3 20.6 21.2 21.1 5 21 20.1 21 21.8 21.1 21.6 21.6 Mean 20.04 20.3420.22 20.9 20.64 20.98 21.44 Standard 0.341467 0.25807 0.376032 0.3114480.299333 0.31209 0.451221 Saline (s.c.) Mouse 1 20 19.8 19.7 20.7 2020.7 21.5 2 19.3 19.2 19.4 20.1 19.8 20.3 20.9 3 20.5 20 20.4 21 20.1 2121.6 4 20.2 20.1 20.3 21 20.2 20.8 21.3 5 20 20.2 20.2 21.1 20.8 21.422.6 6 19 19.2 19.2 20.1 19.6 20.2 20.9 Mean 19.83333 19.75 19.8666720.66667 20.08333 20.73333 21.46667 Standard 0.2319 0.182117 0.206020.18738 0.16816 0.181965 0.256472

TABLE 12 Erythema Days after treatment Mouse 6 7 9 12 13 14 Saline(l.p.) + Irradiation 1 0.5 0 2 5 2 2 2 0 0.5 4 4 3 3 3 0.5 0.5 4 5 5 4 40.5 0.5 1 5 2 3 5 0 0.5 2 5 3 2 Mean 0.3 0.4 2.6 4.8 3 2.8 Standard0.122474 0.1 0.6 0.2 0.547723 0.374166 Saline (s.c.) + Irradiation 1 00.5 2 4 3 2 2 0 0.5 2 5 3 3 3 0 0 2 5 5 3 4 0 0 2 4 2 2 5 0 0.5 1 3 2 2Mean 0 0.3 1.8 4.2 3 2.4 Standard 0 0.122474 0.2 0.374166 0.5477230.244949 Amifostine (l.p.) at 200 mg/kg + Irradiation 1 0.5 0.5 1 4 0.50.5 2 0 0 2 4 2 2 3 0 0 1 3 2 1 4 0.5 0.5 1 3 1 0 5 0 0 1 1 0.5 0 Mean0.2 0.3 1.2 3 1.2 0.7 Standard 0.122474 0.122474 0.2 0.547723 0.3391160.374166 Amifostine (l.p.) at 400 mg/kg + Irradiation 1 0 0 0.5 3 0 0 20 0 1 1 0.5 0.5 3 0 0 0.5 1 0.5 0 4 0 0 1 2 0 0 Mean 0 0 0.75 1.75 0.250.125 Standard 0 0 0.144338 0.478714 0.144338 0.125 Amifostine (s.c.) at200 mg/kg + Irradiation 1 0 0 0.5 2 1 0.5 2 0 0 1 2 0.5 0.5 3 0 0.5 1 12 0.5 4 0 0 3 2 1 0.5 5 0 0 3 3 1 1 Mean 0 0.1 1.7 2 1.1 0.6 Standard 00.1 0.538516 0.316228 0.244949 0.1 Amifostine (s.c.) at 400 mg/kg +Irradiation 1 0 0 2 3 1 1 2 0 0 1 1 0 0 3 0 0 2 1 0.5 0 4 0 0 2 3 1 0 50 0 1 2 1 0.5 Mean 0 0 1.6 2 0.7 0.3 Standard 0 0 0.244949 0.447214 0.20.2 Saline (l.p.) 1 0 0 0 0 0 0 2 0 0 0 0 0 0 3 0 0 0 0 0 0 4 0 0 0 0 00 5 0 0 0 0 0 0 Mean 0 0 0 0 0 0 Standard 0 0 0 0 0 0 Saline (s.c.) 1 00 0 0 0 0 2 0 0 0 0 0 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 5 0 0 0 0 0 0 6 0 00 0 0 0 Mean 0 0 0 0 0 0 Standard 0 0 0 0 0 0

TABLE 13 Oedema Days after treatment Mouse 6 7 9 12 13 14 Saline(l.p.) + Irradiation 1 0.5 0.5 1 2 2 1 2 0 0.5 0.5 2 2 2 3 0.5 0.5 2 2 22 4 0.5 0.5 1 2 2 2 5 0.5 0.5 2 2 2 7 Mean 0.4 0.5 1.3 2 2 2.8 Standard0.1 0 0.3 0 0 1.067708 Saline (s.c.) + Irradiation 1 0 1 2 2 1 2 0.5 1 22 2 3 0.5 0.5 2 2 1 4 0.5 1 2 2 1 5 0.5 2 2 2 2 Mean 0.4 1.1 2 2 1.4Standard 0.1 0.244949 0 0 0.244949 Amifostine (l.p.) at 200 mg/kg +Irradiation 1 1 1 1 2 0.5 0.5 2 0 0 1 2 2 2 3 0.5 0.5 1 2 2 1 4 0.5 0.50.5 1 1 0.5 5 0 0 0.5 0.5 0.5 0 Mean 0.4 0.4 0.8 1.5 1.2 0.8 Standard0.187083 0.187083 0.122474 0.316228 0.339116 0.339116 Amifostine (l.p.)at 400 mg/kg + Irradiation 1 0.5 0.5 0.5 1 0.5 0.5 2 0 0 0.5 0.5 0.5 0.53 0.5 0.5 0.5 0.5 0 0 4 0 0.5 0.5 1 0.5 0.5 Mean 0.25 0.375 0.5 0.750.375 0.375 Standard 0.144338 0.125 0 0.144338 0.125 0.125 Amifostine(s.c.) at 200 mg/kg + Irradiation 1 0.5 0.5 0.5 1 0.5 0.5 2 1 0.5 1 1 11 3 0.5 0.5 1 0.5 1 0.5 4 0.5 0.5 1 1 1 0 5 0.5 0.5 2 1 1 1 Mean 0.6 0.51.1 0.9 0.9 0.6 Standard 0.1 0 0.244949 0.1 0.1 0.187083 Amifostine(s.c.) at 400 mg/kg + Irradiation 1 1 0.5 1 0.5 0.5 1 2 0.5 0.5 0.5 0.50.5 0 3 0.5 0 1 0.5 0.5 0.5 4 1 0.5 1 1 1 0.5 5 0 0.5 1 1 1 0.5 Mean 0.60.4 0.9 0.7 0.7 0.5 Standard 0.187083 0.1 0.1 0.122474 0.122474 0.158114Saline (l.p.) 1 0 0 0 0 0 0 2 0 0 0 0 0 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 50 0 0 0 0 0 Mean 0 0 0 0 0 0 Standard 0 0 0 0 0 0 Saline (s.c.) 1 0 0 00 0 0 2 0 0 0 0 0 0 3 0 0 0 0 0 0 4 0 0 0 0 0 0 5 0 0 0 0 0 0 6 0 0 0 00 0 Mean 0 0 0 0 0 0 Standard 0 0 0 0 0 0

8. EXAMPLE Subcutaneous Administration of Amifostine Stimulated BoneMarrow Cell Growth in Humans

8.1 Subcutaneous Administration of Amifostine in MDS Patients

All patients in the study had MDS and were diagnosed as having one ofthe following subtypes: refractory anemia, refractory anemia with ringsideroblasts, refractory anemia with excess blasts or refractory anemiawith excess blasts in transformation. In addition, the patients hadthrombocytopenia (platelets <100,000/μl), neutropenia (ANC or absoluteneutrophil count<1500/μl) or a hemoglobin untransfused <10 g/dl and/orwere transfusion-dependent as defined by requiring at least 4 units ofRBC in the 10 weeks prior to entry. They had not received any previoustreatment other than transfusion for MDS within 30 days prior to thestudy.

A 500 mg vial of amifostine was dissolved in 2.5 ml of 0.9% NaCl fors.c. administration. If the calculated dose of amifostine required morethan 2 ml volume for dissolution, a total volume was evenly divided andadministered as two s.c. injections. Blood pressure was monitored beforetreatment and after the first dose at 30 minute intervals for up to 90minutes. A single dose of amifostine was given at 500 mg. At least threepatients were treated with each dose. For one cycle of treatment, thepatients were given amifostine once/day Mondays through Fridays forthree weeks, followed by two weeks of rest. Hematologic responses totreatment were evaluated by measuring neutrophil count (ANC), plateletcount, erythroid response including hemoglobin levels and reticulocytecount, and in vitro colony formation.

8.2 Results

Among the patients who received s.c. administration of amifostine, 17patients were evaluated for hematologic responses. Among the 17evaluated patients, 4 previously received amifostine intravenously priorto the study. The patients had diverse cytogenetic patterns with 8patients showing normal karyotype and 9 patients showing abnormalkaryotype. Minimal adverse effects were observed in the treatedpatients: nausea (grade I/II) in one patient, fatigue (grade I/II) infour patients, rash (grade I/II) in six patients, vomiting (grade I/II)in one patient, local reaction (grade III) in one patient and metallictaste (grade I/II) in one patient.

Table 14 shows that 29% of the treated patients showed 50% or greaterincrease of ANC as compared to pre-treatment levels. In addition, 33% ofthe patients showed 50% or greater increase of platelets as compared topre-treatment levels. Moreover, 71% of the patients showed 50% orgreater increase in reticulocyte count as compared to pre-treatmentlevels.

TABLE 14 Evaluable Responders [Magnitude] ANC ≧ 50% ↑ 17  5 (29%)[330-105/μl] ANC < 1000 8 3 (38.5%) ANC > 1000 9 2 (22%) Platelets ≧ 50%↑ 9 3 (33%) [23-32,000/μl] >50,000/μl 3 3 (100%) <50,000/μl 6 0 Reticcount ≧ 50% ↑ 14  10 (71%) [0.4%-4.8%] RBC Transfusion 13  0 (≦50% ↓) BMBlasts ↑ 5 2 (RAEB-t, AML) (RAEB) Hemoglobin 7 1 >1.5 g/dl ↑ RingSideroblast 7 3 ↓ [47%-100%]

Bone marrow progenitor cells were recovered from the treated patientsand assayed for their ability to form CFU-GEMM, BFU-E and CFU-GMcolonies in standard methylcellulose assays. FIGS. 7A-7C shows thatcells obtained from a number of the patients produced bone marrowcolonies. These results indicate that subcutaneous administration ofamifostine in MDS patients was effective in inducing bone marrow cellgrowth.

9. EXAMPLE Subcutaneous Administration of Amifostine and RadiationTherapy in Patients with Head and Neck Cancer

9.1 Subcutaneous Administration of Amifostine in Patients with Head andNeck Cancer

The advantages of subcutaneous (sc) administered amifostine wasdemonstrated by comparing the results of a phase II trial ofsubcutaneously administered amifostine and radiation therapy in head andneck cancer patients with the results of a study wherein the patientsreceived IV amifostine and radiation therapy for head and neck cancer(Brizel, J Clin Oncol 18:3339-3345, 2000).

In the phase II study all patients were undergoing radiation therapy forhistologically confirmed squamous cell carcinoma of the head and neckregion. Patients were allowed to enter the study following surgery withcurative intent as soon as the wound has healed but no later than 12weeks post operatively. Patients were at least 18 years of age with anexpected survival rate of at least 12 months, a Karnofsky performancestatus of at least 70, no evidence of a distant metastatic disease, hada granulocyte count (segs and bands) of at least 2000/mm³ and a platletcount of at least 100,000/mm³, a serum creatinine of less than 2.0mg/dL, a total bilirubin of less than 2.0 mg % and a SGOT of 3 times orless the upper limit of normal, had at least 75% of each parotid glandin the treatment fields that received a minimum dose of 40 Gy, and werenot entered in any other investigational therapeutic trials.

Patients were excluded if they had a primary lesion of the parotidgland, would be receiving hyperfractionated or accelerated radiotherapy,had a history of prior malignancies within the past 5 years other thannon-melanomatous skin cancers that had been controlled or carcinoma insitu of the cervix, were receiving concurrent chemotherapy (patients mayhave received chemotherapy for this head and neck cancer more than threeweeks prior to enrollment), were concurrently using pilocarpine, hadbeen treated with any investigational drugs more than 4 weeks prior toentering the study, had general or psychological conditions that wouldnot permit the patient to complete the study, or were women of childbearing potential not using an effective method of birth control.

Patients received amifostine as two 250 mg subcutaneous injections (1.25mL each) into two locations on the abdominal wall 60 minutes (±15minutes) prior to radiation therapy (1.8-2.0 Gy/day for 25-35fractions). Patients were hydrated with 250 cc of fluid orally 30minutes prior to amifostine to assure that they were not dehydrated orhave a decreased vascular volume. Patients were kept in a supineposition during amifostine administration and for 10 minutes followingamifostine administration. Blood pressure and pulse were obtained priorto subcutaneous administration and repeated 10 minutes followingadministration. An IV was available to treat hypotension if it occurred.Patients received full supportive care including antiemetics,antibiotics, transfusions of blood and blood products, and the like whenneeded. Prophylactic antiemetics were not administered. If patientsexperienced nausea and vomiting they were administered eitherprochlorperazine, 10 mg PO, 30 minutes prior to therapy, repeating every4 to 6 hours following therapy as needed or ondansetron, 8 mg PO, 30minutes prior to therapy, repeating every 4 hours following therapy asneeded.

Radiation therapy was provided with linear accelerators with appropriatephoton and electron boosting to the nodes or Cobalt 60. The appropriatephoton energy was based on optimizing the radiation therapy dosedistribution within the target volume and minimizing dose to normaltissue. Patients were reproducibly mobilized and radio-opaque markerswere used to delineate the extent of nodal disease and, where possible,the primary tumor. All fields were treated once daily at 1.8-2.0 Gy perfraction, five days per week to a total dose of:

Post operative low risk patients: 50-60 Gy (primaries with negativetumor margins, node negative, node positive without extra capsularextension);

Post operative high risk patients: 60-66 Gy (positive tumor margins, N2,N3; any extra capsular extension in the neck); and

Definitive radiation patients: 66-70 Gy. Electrons were used to boostthe nodal regions in the posterior neck when additional treatment tothese regions was indicated. Fields were reduced to exclude the spinalcord at 40-46 Gy at the midplane. The entire neck, however, wasirradiated to a minimum dose of 46 Gy (even at stage No) at anatomicallevels of lymph node spread usually 2-4 cm below the skin surface.Positive neck nodes received a minimum dose of 60 Gy in 30-35 fractionsin 6-7 weeks. To supplement the dose to the posterior neck and cinicallypositive nodes, boost techniques included additional electron beam (>6MeV) to the posterior neck, wedge pair or oblique fields. Initially,clinically negative posterior neck nodes received a minimum dose of44-46 Gy at 3 cm depth.

9.2 Results

Xerostomia was graded in each subject according to the RTOG criteria. 55patients were enrolled, with 54 evaluable for xerostomia. Results areshown in Table 15, along with similar results from a phase III trial inwhich 200 mg/m² of amifostine was administered as a 3 minute IV infusion15 to 30 minutes before radiation therapy (1.8-2 Gy/day, 54-70 Gytotal)(Brizel, J Clin Oncol 18:3339-3345, 2000).

TABLE 15 Sc IV Amifostine + Amifostine RT RT p-value N 54 148 153 ≧Grade2 acute xerostomia Incidence All patients 56% 51% 78% <0.0001 Patients49% 51% 78% <0.0001 receiving ≧60 Gy RT Median time to 40 days 45 days30  0.0001 onset days Median cumulative 50 Gy 60 Gy 42 Gy  0.0001 RTdose to onset

Acute xerostomia, i.e., >grade 2 on the RTOG scale, was observed inpatients administered subcutaneous amifostine at a comparable rate tothose that were administered IV amifostine. There were, however, noreports of Grade 3 hypotension or Grade 3 nausea/vomiting following SCamifostine (3% and 7%, respectively, with IV). Grade 3 generalizedcutaneous toxicity occurred in 13% of patients (3% with IV). The resultsof this study demonstrate that subcutaneous (SC) amifostine, similar toIV amifostine, protects against radiation-induced xerostomia but reducesor avoids side effects known to occur with IV amifostine The data showsthat SC amifostine provides comparable protective effects againstradiation-induced xerostomia and reduces the amifostine-related sideeffects of nausea, vomiting, and hypotension in head and neck cancerpatients.

10. EXAMPLE Extended Window of Mucosal Radioprotection of Intravenous orSubcutaneous Amifostine in Rats

9.2 Experimental Design

A rat RT model was used to examine the protective effects of amifostinefollowing IV as well as subcutaneous (SC) administration, and toinvestigate the length of time between amifostine administration and RT.In these experiments, rats were given 200 mg/kg (1200 mg/m²) ofamifostine IV or SC, and their head and neck regions exposed to 15.3 Gyof gamma irradiation at various times following amifostineadministration. For 10 days after treatment, the rats were weighed andthe oral cavities of the rats were examined for signs of mucositis.Mucosal erythema and mucosal edema were scored according to 0-5 and 0-2scales, respectively, with the scores added to indicate overallmucositis.

10.2 Results

The results for IV amifostine after 10 days are summarized in Table 16.

Pretreatme nt interval Overall Group (hr.) Erythema Edema Mucositis RTNA* 2.0 1.4 3.4 RT + 0.5 0 0 0 amifostine RT + 2 0 0 0 amifostine RT + 40 0 0 amifostine RT + 8 2.8 1.2 4.0 amifostine *(NA = not applicable).

At 200 mg/kg (1200 mg/m²), SC administration of amifostine gaveradioprotection comparable to IV administration up to 4 hours prior toradiation therapy.

The present invention is not to be limited in scope by the exemplifiedembodiments, which are intended as illustrations of individual aspectsof the invention. Indeed, various modifications for the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

All publications cited herein are incorporated by reference in theirentireties for all purposes.

What is claimed is:
 1. A method of protecting against or treating thetoxicities associated with ionizing radiation while reducing sideeffects in a subject comprising: (a) subcutaneously administering to asubject in need thereof within about 3 hours before the subject isexposed to ionizing radiation an effective amount of a compound havingthe formula: R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I)  or a pharmaceuticallyacceptable salt or hydrate thereof, wherein: R₁ is hydrogen, C₆-C₇ aryl,C₂-C₇ acyl, or C₁-C₇ alkyl; R₂ is hydrogen or PO₃H₂; n is an integerfrom 2 to 6; and m is an integer from 2 to
 6. 2. The method of claim 1,wherein the compound is administered between about 20 minutes to 90minutes before the subject is exposed to the ionizing radiation.
 3. Themethod of claim 1, wherein the compound is administered about 1 hourbefore the subject is exposed to the ionizing radiation.
 4. The methodof claim 1, wherein the toxicity is one or more selected from the groupconsisting of neurotoxicity, nephrotoxicity, ototoxicity,cardiotoxicity, alopecia, mucositis, xerostomia, infertility, pulmonarytoxicity, and renal failure.
 5. The method of claim 4, wherein thetoxicity is xerostomia.
 6. The method of claim 5, wherein the toxicityis acute xerostomia.
 7. The method of claim 5, wherein the toxicity islate xerostomia.
 8. The method of claim 4, wherein the toxicity ismucositis.
 9. The method of claim 8, wherein the toxicity is acutemucositis.
 10. The method of claim 8, wherein the toxicity is latemucositis.
 11. The method of claim 1, wherein amount administered is atleast about 500 mg.
 12. The method of claim 11, wherein the amountadministered is at least about 500 mg to 1500 mg.
 13. The method ofclaim 12, wherein the compound is S-2-(3-aminopropylamino)ethyldihydrogen phosphorothioate or S-3-(3-methylaminopropylamino)propyldihydrogen phosphorothioate.
 14. The method of claim 13, wherein thecompound is S-2-(3-aminopropylamino)ethyl dihydrogen phosphorothioate.15. The method of claim 1, wherein the compound is administered as twosubcutaneous injections.
 16. The method of claim 1, wherein the compoundis administered in the form of a pharmaceutical composition comprisingthe compound and a pharmaceutically acceptable diluent.
 17. The methodof claim 16, wherein the diluent is normal saline.
 18. The method ofclaim 1, wherein the compound is S-2-(3-aminopropylamino)ethyldihydrogen phosphorothioate in an amount of about 500 mg to 1500 mg. andis administered to the subject about 1 hour before the subject isexposed to the ionizing radiation.
 19. The method of claim 18, whereinthe amifostine is administered as two subcutaneous injections.
 20. Themethod of claim 1, wherein the subject is a mammal.
 21. The method ofclaim 18, wherein the mammal is a human.
 22. A method of treating headand neck cancer with radiation therapy comprising subcutaneouslyadministering to a subject in need thereof within about 3 hours beforethe subject is exposed to ionizing radiation an effective amount ofamifostine.
 23. The method of claim 22, wherein the radiation isadministered once daily at a dose of about 1.8 to 2.0 Gy per fractionfor a total dose of 50 to 70 Gy.
 24. The method of claim 22, wherein theamount administered is at least about 500 mg.
 25. The method of claim24, wherein the amount administered is at least about 500 mg to 1500 mg.26. The method of claim 22, wherein the amount is administered as twosubcutaneous injections.
 27. The method of claim 22, wherein thecompound is administered in the form of a pharmaceutical compositioncomprising the compound and a pharmaceutically acceptable diluent. 28.The method of claim 27, wherein the diluent is normal saline.
 29. Amethod of preventing xerostomia or mucosities in a subject receivingradiation therapy comprising: (a) subcutaneously administering to asubject in need thereof within about 3 hours before the subject isexposed to ionizing radiation an effective amount of a compound havingthe formula: R₁NH(CH₂)_(n)NH(CH₂)_(m)SR₂  (I)  or a pharmaceuticallyacceptable salt or hydrate thereof, wherein: R₁ is hydrogen, C₆-C₇ aryl,C₂-C₇ acyl, or C₁-C₇ alkyl; R₂ is hydrogen or PO₃H₂; n is an integerfrom 2 to 6; and m is an integer from 2 to
 6. 30. The method of claim29, wherein patient is a cancer patient.
 31. The method of claim 30,wherein the cancer is head and neck cancer.
 32. The method of claim 29,wherein the amount administered is at least about 500 mg.
 33. The methodof claim 32, wherein the amount administered is at least about 500 mg to1500 mg.
 34. The method of claim 29, wherein the amount is administeredas two subcutaneous injections.
 35. The method of claim 29, wherein thecompound is administered in the form of a pharmaceutical compositioncomprising the compound and a pharmaceutically acceptable diluent. 36.The method of claim 35, wherein the diluent is normal saline.
 37. Themethod of claim 18, wherein the S-2-(3-aminopropylamino)ethyl dihydrogenphosphorothioate is administered to the subject about 1 hour before thesubject is exposed to the ionizing radiation.
 38. The method of claim 1,wherein the compound of formula (I) is S-2-(3-aminopropyl)ethyldihydrogen phosphorothioate.
 39. The method of claim 29, wherein thecompound of formula (I) is S-2-(3-aminopropyl)ethyl dihydrogenphosphorothioate.